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.