DNA methylation of ERAP1 gene
In total, two CpG islands containing 31 loci were tested the proportion of methylated cytokine accounting for the total cytokine. Among the 15 loci of ERAP1_1 island and 16 loci of ERAP2 island, 13 CpG sites, ERAP1_1_46, ERAP1_1_91, ERAP1_1_107, ERAP1_1_118, ERAP1_1_222, ERAP1_2_28, ERAP1_2_31, ERAP1_2_46, ERAP1_2_49, ERAP1_2_69, ERAP1_2_95, ERAP1_2_121 and ERAP1_2_136, were significant hypermethylated in AS patients compared to HCs used Mann-Whitney U test detailed in Table 2. Logistic regression also indicated that methylation of six loci in ERAP1_2 island, ERAP1_2_28, ERAP1_2_31, ERAP1_2_49, ERAP1_2_69, ERAP1_2_121 and ERAP1_2_136, were significantly associated with increased risk of AS visualized in Figure 1A. Among them, ERAP1_2_28 located at chr5: 96143595 was reported to have highest effect size with OR = 5.390 (95% CI: 2.048 to 14.184) of as a percent of total cytokine methylated. The average methylation levels of ERAP1_1 (Z= -4.831, P < 0.001) and ERAP1_2 (Z = -6.140,P < 0.001) islands were also calculated to associated with AS (Figure 1B & 1C). ROC curve analysis on the average methylation levels of the two CpG islands also indicated their potential as biomarkers of AS. The AUC of ROC curve for ERAP1_1 island was 0.669 (95% CI: 0.626 to 0.707, P < 0.001), with a sensitivity of 0.818 and a specificity of 0.531 for the best cutoff point 0.671 (Figure 1D, Table 3). Correspondingly, the AUC of ERAP1_2 was 0.750 (95% CI: 0.683 to 0.818,P < 0.001), and the best cutoff point was 0.681, with a sensitivity of 0.707 and specificity of 0.684 (Figure 1D, Table 3). We also established a regression model included the above mentioned 13 significant CpG loci detailed in Table S1, with each sample obtained a probability value. ROC curve analysis on the probability value also reported that the AUC was 0.779 (95% CI: 0.714 to 0.844, P< 0.001), and best cutoff point was 0.500, with a sensitivity of 0.717 and specificity of 0.737 (Figure 1D, Table 3).