Study populations
A total of 100 AS patients and 100 age and gender matched HCs were enrolled in the DNA methylation examination stage, and 20 patients and 20 controls were recruited in the mRNA expression verification stage. All patients and HCs were recruited from the Department of Rheumatology at the First Affliation Hospital of Anhui Medical University. Diagnosis of AS was made by qualified rheumatologists according to the modified New York criteria (20). Blood donors with no history of rheumatic diseases or other chronic diseases were included as controls. DNA and mRNA samples were extracted from the 5 ml peripheral venous blood of all participants. Besides, all patients have filled out a questionnaire about the general geographic and clinical characteristics. Detailed clinical indicators as medication use, HLA-B27, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Bath Ankylosing Spondylitis Functional Index (BASFI) were reported. This study was approved by the ethics committee of Anhui Medical University with the serial number of 20170225. All patients and controls provided their written informed content.
Targeted bisulfite sequencing assay
CpG islands in the proximal promoter of ERAP1 gene were examined from 2k bp upstream of transcript start site to 1k bp downstream of the first exon satisfying the following criteria: a) observed to expected ratio of CpG dinucleotide > 0.60; b) percentage of cytosine and guanine > 50%; c) length >200bp. DNA sequences of the CpG islands in ERAP1 promoter region were determined by an online database of the University of California, Santa Cruz (http://www.genome.ucsc.edu), and the primers sequences for ERAP1 methylation were designed by the EpiDesigner online software (http:// www.epidesigner.com) accordingly. And 31 methylation sites of two CpG islands (ERAP1_1 and ERAP1_2) were analyzed in our study. The primers sequence of ERAP1_1 and ERAP1_2 methylation were listed as following: ERAP1_1, forward: 5’- AGGGTTAGGGGTATGTAGGAAAG-3’, reverse: 5’- CCTTCCTCCTCTACAACATCTCC-3’; ERAP1_2, forward: 5’- GTTTTGGGGTYGTTTTTATTTTTG-3’, reverse: 5’- TTACCCTTTCCCCAACTCC -3’.
Genomic DNA was firstly extracted from peripheral venous blood of participants using DNeasy Blood Tissue Kit QIAGEN Kit (QIAGEN, Germany) in line with manufacturer’s protocol, and quantified and qualified through NanoDropTM2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA.). Genomic DNA (400 ng) was bisulfite converted using EZ DNA MethylatioTM-GOLD Kit (Zymo Research, Irvine, USA). Multiplex polymerase chain reaction (PCR) was performed with above primers combination, and PCR amplicons were separated and purified through agarose electrophoresis and QIAquick Gel Extraction kit (QIAGEN, Germany). Corresponding libraries were sequenced on Illumina NextSeq platform according to manufacturer’s protocols.