DDH patients and healthy controls
DDH patients were consecutively recruited between 2017 and 2018 from the orthopedics clinic of Imam Khomeini Hospital of Tehran University of Medical Sciences. The healthy subjects are participated in the study at the same time. Diseases such as osteoarthritis, metabolic bone disease and DDH were excluded from the control group. Our study comprises of 90 individuals (45 healthy controls and 45 DDH patients), and the mean age ± SD of controls and patients was 42 ± 15.2 and 45 ± 12.6, respectively. The male-to-female ratio was equal in patient and control groups (5 males and 40 females). The control individuals had neither family history nor clinical evidence of arthritis or any inflammatory disorders. The control samples were taken from people who were under a hemi- or total arthroplasty operation because of femoral head fracture. These cartilage samples are actually obtained from individuals with healthy femoral head. The Human Research Ethics Committee of Tehran University of Medical Sciences approved this study (IR.TUMS.IKHC.REC.1397.179). Informed consent was obtained from all control and patient subjects.
Sample collection
Samples were collected from the femoral head cartilage of healthy control subjects and DDH patients. QIAamp DNA Mini Kit (Qiagen) was used to extract genomic DNA from the samples according to the manufacturer’s instruction. DNA from all the samples extracted and stored at − 20 °C. Purity and yield of DNA were determined using a NanoDrop spectrophotometer at 260 nm and 280 nm (NanoDrop 2000c Spectrophotometer, Thermo Fisher Scientific, Wilmington, DE, USA).
Bisulfite treatment
The extracted DNA was treated with bisulfite (EpiTect Plus DNA Bisulfite Kit). The principle of this method of treatment is that the methylated cytosines will remain unchanged, while the unmethylated cytosine will be converted to uracil. According to bisulfite treatment kit, the following notes were applied before starting.
30 ml of 96% ethanol was added to Buffer BW and stored at room temperature (15–25 °C). 27 ml of 96% ethanol is added to buffer BD and stored at 2–8 °C. 310 μl of RNase-free water is added to carrier RNA and stored at − 20 °C.
EpiTect Plus DNA Bisulfite Kit
800 μl of RNase-free water was added to each aliquot of Bisulfite Mix and then vortexed to completely dissolve the Bisulfite Mix (several minutes). 200 μl PCR tubes were used for bisulfite reactions based on the protocol. The tubes were closed and mixed for several minutes. The blue color of the DNA Protect Buffer is considered as a correct PH and sufficient mixture. Thermal cycler program was set up, the tubes were located in the thermal cycler, and the incubation time for each section was obtained from the protocol.
After PCR, the tubes were centrifuged and the reactions were transferred into microcentrifuge clean tubes (1.5 ml), and then, 310 μl of Buffer BL was added into each tube according to the protocol, mixed and centrifuged shortly. Afterward, 250 μl of 96% ethanol was added into each tube, mixed, vortexed and centrifuged.
Then, the reactions from tubes were transferred into spin columns which were located on collection tubes and centrifuged for 60 s. The flow-through was discarded from collection tubes, and spine tubes were placed on collection tubes again.
The Buffer BW (500 μl) was added into each spine column, and the tubes were centrifuged for 60 s. Again, the flow-through was discarded from collection tubes and spine tubes were placed into collection tubes.
The Buffer BD (500 μl) was added into each spin column and incubated at room temperature for 15 min and then centrifuged for 60 s. Afterward, the flow-through was discarded from collection tubes and spine tubes were placed into collection tubes.
The Buffer BW (500 μl) was added into each spin column and centrifuged for 60 s. Then, the flow-through was discarded from collection tubes and spine tubes were placed into collection tubes. (This step was repeated again.)
The 96% ethanol (250 μl) was added into each spin column and centrifuged for 60 s. Then, spin columns were placed into collection tubes (2 ml) and centrifuged for 60 s. In order to evaporate the liquid, the tubes were incubated on a heating block for 5 min at 60 °C.
The spin columns were placed into microcentrifuge clean tubes (1.5 ml). Then, the Buffer EB (15 μl) was added into each spin column, incubated at room temperature for 60 s. To elute the DNA, tubes were centrifuged. Finally, the purified DNA was stored for up to 24 h at 2–8 °C. In order to use samples for more works and further evaluation, DNA should be stored at − 20 °C.
PCR amplification
A 221 base-pair segment of DNA at 3p22 band was target for PCR amplification. The segment was part of the promoter region of CX3CR1 gene. The DNA sequence of CX3CR1 promoter was obtained from the UCSC (University of California, Santa Cruz) website (https://genome.ucsc.edu/). The bisulfite specific primers for PCR were designed using MethPrimer website (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi).
Each PCR tube contained DNA Protect Buffer (35 μl), bisulfite mix (85 μl), RNase-free water (variable) and bisulfite-treated DNA (maximum 20 μl). For samples with high concentration (ranged from 1 ng to 2 μg), the final volume of RNase-free water and DNA solution was 20 μl. The PCR conditions were as follows: denaturation (95 °C for 5 min), incubation (60 °C for 25 min), denaturation (95 °C for 5 min), incubation (60 °C for 85 min), denaturation (95 °C for 5 min) and incubation (60 °C for 175 min). For amplification validation, all DNA samples were gel electrophoresed after PCR amplification with 2% agarose gel. After confirmation with electrophoresis, the samples were sequenced (Macrogen, Seoul, Korea) and evaluated using Codon Code Aligner version 2 (Codon Code Corporation, Dedham, MA, USA) software.
Statistical analysis
Our data were analyzed with SPSS version 22.0, and graph was generated using GraphPad Prism version 5 for windows (GraphPad Software, La Jolla, CA USA, www. graphpad.com). All data are shown as mean ± standard deviation (S.D.) and also analyzed for normal distribution using the Kolmogorov–Smirnov test. Mann–Whitney test and independent sample t test were applied. Value less than 0.05 was considered statistically significant.