Table 2.
Summary of latent DNA methylation axes associated with diagnosis and symptom burden
| Feature | PC5 | PC6 |
|---|---|---|
| Main symptom associations | Post-exertional malaise and cognitive symptoms | Pain impact, widespread pain, temporal summation, sleep disturbance, post-exertional malaise, and cognitive symptoms |
| Diagnostic association | Differentiated ME/CFS from fibromyalgia and healthy controls | Differentiated both patient groups from healthy controls, but not ME/CFS from fibromyalgia |
| Main clinical pattern | ME/CFS-aligned axis | Multisystem pain and symptom-burden axis |
| High-confidence DMRs | 591 regions | 54 regions |
| Overlap with group-associated DMRs | Strongest overlap with ME/CFS-associated regions | Smaller diagnostic overlap; broader symptom association profile |
| Selected loci | OXT, HIF3A, FAM171A2, NAPRT, SPRED3 | FURIN, PDCD1, HIF3A, ESR2/SYNE2, FAM171A2, PREX1 |
| Main enriched pathways | Neuroactive ligand signalling, calcium signalling, PI3K-Akt, Wnt, PPAR, arachidonic acid metabolism | Cytokine-cytokine receptor interaction, NF-κB, JAK-STAT, TGF-β, Notch signalling |
| Robustness | Stable across bootstrap, permutation, leave-one-out, immune-cell, medication, and comorbidity sensitivity analyses | Visceral symptom association was more sensitive in leave-one-out and medication/comorbidity analyses; widespread pain association attenuated after opioid adjustment but remained statistically significant |
| Interpretation | Methylation axis associated with PEM and cognitive symptoms, and ME/CFS diagnosis | Methylation axis associated with broader symptom burden across ME/CFS and fibromyalgia, with a particular relevance for pain-related symptoms |
PC5 and PC6 were identified using a-priori unsupervised principal component analysis of processed PBMC DNA methylation data. Diagnostic and symptom associations were tested using linear models adjusted for age, body mass index, and estimated immune-cell composition. DMRs were retained using predefined region-level filtering criteria. Robustness was assessed using bootstrap resampling, permutation testing, leave-one-out analyses, additional immune-cell adjustment, and medication/comorbidity sensitivity analyses. DMR indicates differentially methylated region; ME/CFS, myalgic encephalomyelitis/chronic fatigue syndrome; PBMC, peripheral blood mononuclear cell; PEM, post-exertional malaise.