Abstract
BACKGROUND: Headache disorders are among the leading causes of neurological disability worldwide, affecting over half of the global population. Recent data indicate that headache prevalence among young people increased by 23.7% between 1990 and 2021. Migraine, a highly disabling primary headache, significantly impacts daily life, yet timely and accurate diagnosis remains challenging. Many patients self-manage with analgesics, contributing to medication-overuse headache (MOH), which affects 1–2% of the general population. Emerging evidence suggests that epigenetic mechanisms, particularly DNA methylation (DNAm), play a key role in the pathophysiology of migraine and MOH. METHODS: We investigated epigenetic entropy by calculating Stochastic Epigenetic Mutations (SEMs) and epivariations in 57 participants of the longitudinal Epimode project. Patients were enrolled in three groups: Chronic Migraine with MOH (assessed before and at various timepoints after clinical treatment), Episodic Migraine (EM; assessed at baseline and follow-up), and Healthy Controls (HC; assessed at baseline and follow-up). Peripheral blood DNA methylation was analysed genome-wide, and SEMs were annotated by genomic context and functional category. Statistical analyses assessed group differences and temporal dynamics of SEMs. RESULTS: At baseline (T0), both EM and MOH patients exhibited significantly higher SEMs counts compared with HC, with no differences between EM and MOH groups, either in total counts or in hypo-/hypermethylated CpGs. Differences persisted across functional categories and chromosomes. Overall, no significant changes in SEMs counts were observed in MOH patients pre- and post-treatment overall. However, MOH non-responders displayed high SEMs counts at T0 and the greatest reshuffling rate between lost, maintained and gained SEMs over time. Enrichment analyses identified SEMs in genes associated with metabolic, synaptic, and inflammatory function. CONCLUSION: These findings indicate that migraine promotes epigenetic entropy and suggest that epigenetic instability contributes to headache chronification and treatment resistance. Moreover, the dynamics of SEMs over time provide more informative insights than absolute counts alone in evaluating clinical outcomes. This study reinforces the relevance of epigenetic mechanisms in the clinical management of headache disorders. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at10.1186/s10194-026-02343-w.