Expression level and correlation analysis of ERAP1 mRNA
In order to verify the differential methylation level of ERAP1 , we also measured the mRNA expression level in 20 AS patients and 20 HCs. The expression level of ERAP1 was significant decreased in AS patients compared with HCs ((Z = -4.048, P < 0.001, Figure 1e). In the correlation analysis, we also found that body mass index (rs = -0.659, P = 0.002), chest expansion (rs = 0.697, P = 0.001) and Schober test score (rs = 0.537, P = 0.018) were associated with ERAP1 level.
Discussion
Our study first proved that the relationship betweenERAP1 and AS from the epigenetic aspect. The result proved that hypermethylation ofERAP1 promoter in the peripheral blood was associated with AS. The function and disease activity index and NSAIDs were also significantly associated with the methylation level. Correspondingly, the mRNA expression level was also proved to significantly decrease in AS patients.
Unlike the DNA text, the sequence of nucleotides, containing the genetic information, the annotation system of chemical modification was exited for instructing how and when to read the text in mammals. DNA methylation plays sophisticated roles in annotating genetic information. The existence of DNA methylation in gene regulatory regions, as promoter and enhancer, would recruit a group of factors programmed generating a closed chromatin structure and consequently repressing the gene expression. Our study reported that the DNA methylation of ERAP1was associated with AS, consistent with results of previous studies thatEARP1 plays pivotal roles in the pathogenesis of AS. In the verification stage, we also found that the mRNA expression level of AS patients were significantly deceased, which is consistent with the result that ERAP1 gene was hypermethylated in AS patients.
Different form the sequence of DNA, previous study has proved that DNA methylation patterns derived from the gametes will erased before embryo implantation and the new methylation profile will established in each mammal (21, 22). Intriguingly, we found something unusual that ERAP1_1 in AS patients with family history were hypomethylated. One reason may be that patients with family history inherited higher risk of AS, and the pathogenesis of AS propositus was more like to the results of exposure of environmental risk factors, which made them have higher methylation level than patients with higher inherited risk of AS. Even though ERAP1 was established to cooperate with HLA-B27 in AS, the results indicated that HLA-B27 status may not associated with the methylation level of ERAP1 in AS patients. Consistently, previous studies also indicated that family history was associated with methylation level of diseases related genes (23, 24). Our study also suggested that NSAIDs was associated with the methylation of ERAP1_1, and the tumor necrosis factor inhibitor and sulfasalazine were irrelevant with the methylation level. Similarly, previous study also proved that celecoxib could reverse the DNA hypomethylation status in rat colon tumors. This study also indicated that preventive efficacy of various agents may be the results of their effect on reverse DNA hypomethylation in some extent (25). Our results also indicated that the efficacy of NSAIDs on repression the progression of ossification may be the results of epigenetic regulation of corresponding genes. However, the nature of cross-sectional design our study make us only can provide association result. The result should be verified by prospective study, and further study about the underlying mechanism was also helpful.
In the correlation analysis, we also found that the methylation level of ERAP1_2 was positively associated with the X-ray classification of sacroiliac joint. Besides, we also found that the mRNA level ofERAP1 was positively associated the thoracic and lumbar mobility. It seemed that ERAP1 was more associated with the long-standing joint ossification. And the higher methylation and corresponding lower mRNA levels of ERAP1 was associated with severer ossification. These results were consistent with the conclusion that ERAP1 were associated with AS. Besides, we also found something interesting that body mass index was positively associated with the mRNA level ofERAP1 . Relevant study was still devoid, and the relationship should be verified in further with larger sample size. The ROC curve analysis indicated that the ERAP1 methylation level could serve as biomarkers, by either CpG island or entire promoter, to distinguish AS patients from HCs.
Our study has several strengths. To best of our known, this study was the first research reported the relationship between ERAP1methylation status and AS and verified the mRNA expression level ofERAP1 . Besides, we have also evaluated the relationship between various environmental factors, clinical manifestations or medication use and the methylation level. Considering the less invasiveness and higher diagnostic efficiency, methylation test was valuable in clinical setting for AS diagnosis. Recent study indicated that drugs as celecoxib was could prevent diseases as tumor (25), which suggested the treatment potential of agents specially regulated the methylation factors ofERAP1 . The finding that NSAIDs use was positive associated the methylation level of ERAP1_1 also indicated the treatment efficacy of NSAIDs on AS was via altering the methylation level of corresponding genes in some extent.
These are also some limitation should be considered. First, subjects for the testing of DNA methylation and mRNA were two separate groups, so the relationship analysis between DNA methylation and mRNA was not applicable. Second, the methylation profiles were different across tissues and cell subtypes. ERAP1 methylation level of peripheral blood mononuclear cells can not present the methylation of specific cell. In the end, the single center case-control study design limited the generalizability of results, and further lager scale prospective study and animal model research are necessary.