DNA-methylation and immunological response in medication overuse headache

Louise Ninett Carlsen, Christine Søholm Hansen, Lisette J A Kogelman, Thomas Mears Werge, Henrik Ullum, Jonas Bybjerg-Grauholm, Thomas Folkmann Hansen, Rigmor Højland Jensen


OBJECTIVE: To investigate whether medication-overuse headache patients have differential DNA-methylation pattern.

METHODS: We collected blood samples from 120 medication-overuse headache-patients, 57 controls (29 episodic migraine patients and 28 healthy controls) in a hypothesis-generating cross-sectional case-control pilot study; 100 of the medication-overuse headache-patients were followed for six months and samples were collected at two and six months for the longitudinal methylation analyses. Blood cell proportions of leucocytes (neutrophils, NK-cells, monocytes, CD8+ and CD4+ T-cells, and B-cells) and the neutrophile-lymphocyte ratio were estimated using methylation data as a measure for immunological analysis and a cell type-specific epigenome wide association study was conducted between medication-overuse headache-patients and controls, and longitudinally for reduction in headache days/month among medication-overuse headache-patients.

RESULTS: We found a higher neutrophile-lymphocyte ratio in medication-overuse headache-patients compared to controls, indicating a higher immunological response in medication-overuse headache-patients (false discovery rate (adjusted p-value)<0.001). Reduction in headache days/month (9.8; 95% CI 8.1-11.5) was associated with lower neutrophile-lymphocyte ratio (false discovery rate adjusted p-value = 0.041).Three genes (CORIN, CCKBR and CLDN9) were hypermethylated in specific cell types in medication-overuse headache-patients compared to controls. No methylation differences were associated with reduction in headache days in medication-overuse headache-patients after six months.

CONCLUSION: This pilot study was consistent with higher immunological response in medication-overuse headache-patients which decreased with a reduction in headache days in longitudinal analysis. medication-overuse headache-patients exhibited differential methylation in innate immune cells but did not exhibit longitudinal differences with alterations in headache days. Our study creates hypotheses for further biomarker searches.ClinicalTrials.gov Identifier: NCT02993289.

Original languageEnglish
JournalCephalalgia : an international journal of headache
Issue number3
Pages (from-to)3331024221147482
Publication statusPublished - Mar 2023


  • Humans
  • Cross-Sectional Studies
  • Pilot Projects
  • Headache Disorders, Secondary/genetics
  • Migraine Disorders/drug therapy
  • Headache


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