Fig. 2

Schematic representation of the analysis. TS1 and TS2 denote the tests comparing ACPA-positive healthy twin versus ACPA-negative healthy twin and ACPA-positive RA twin versus ACPA-negative healthy twin, respectively. Steps 1 and 2 denote the first step of the analysis, DMR identification in TS1 and TS2 without cell proportion adjustment. In step 3 we computed DMRs between each pair of cell types (neutrophils, CD4⁺ T cells, CD8⁺ T cells, and CD56⁺ NK cells) and observed that DMRs identified without cell proportion adjustment were associated with cell type, so likely to be associated with changes in cell proportion. For this reason in step 4 we estimated cell proportion in each sample by adapting the method of Houseman et al. [31] (see the “Cell proportion estimation” section in the “Methods”). In step 5 and 6 we used cell proportion estimations as covariates in the identification of DMRs in TS1 and TS2. A DMR is considered statistically significant if it is significant both globally (FWER <0.10) and locally (permuted p-value <0.10); details are provided in the “Methods”