This experiment was approved by the Ethics Committee of our Hospital (No.201703358) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all patients and controls who participated the experiments and from their legal guardians.
Subjects and samples
All patients were those who received treatment at the our hospital between September 2016 and September 2018. In the first discovery stage, there were two pairs of monozygotic twins. In the second methylation of the target region stage, 50 CS and 50 control were enrolled in the experiment. And in the third gene expression stage, 20 CS and 20 control specimens were collected for the validation. The inclusion criteria for the CS and control group were as follows: children who signed the informed consent; complete clinical data and imaging data; first diagnosed with CS in our hospital based on medical history, physical examination, and auxiliary examination without any treatment before; and belonged to the Chinese Han population. Since the immunohistochemistry samples were surgically obtained, and children younger than 10 who undergo spinal surgery but are not patients with spinal deformities are rare, patients aged 10 to 18 years with lumbar disc herniation, thoracolumbar fractures, and spondylolisthesis were selected as controls. Patients with CS who were 10 to 18 years old were selected as the case group for the immunohistochemical stage. The exclusion criteria for both groups included the lack of informed consent, a clear history of the mother taking medication or toxic substances during the pregnancy, incomplete data of the child patient, history of treatment with a spinal brace, and the presence of any of various syndromes. Detailed information was listed in Table 1.
Agilent SureSelect XT Human Methyl-Sequencing(14)
DNA was extracted from peripheral blood using the SQ Blood DNA Kit II (OMEGA Bio-TEK, USA). For the Target Bisulfite Sequencing library constructing, we use NimbleGen SeqCap Epi Enrichment System to capture the target genomic regions. The experiment contains several step：(1) Genomic DNA: NimbleGen SeqCap Epi oligo pool is made against target regions in the genome.(2) Library Preparation: Standard shot-gun sequencing library, with methylated adapters, is generated from genomic DNA.(3) Bisulfite Conversion: The sequencing library is bisulfite treated to convert unmethylated cytosines to uracil.(4) Hybridization: The bisulfite treated sequencing library is hybridized to the SeqCap Epi oligo pool.(5) Bead Capture: Capture beads are used to pull down the complex of capture oligos and genomic DNA fragments.(6) Washing: Unbound fragments are removed by washing.(7) Amplification: Enriched fragment pool is amplified by PCR.(8) Bisulfite sequencing-ready DNA: Enriched fragment pool is amplified by PCR.
Putative Different Methylation Regions in gene promoters were identified by comparison of the case and control methylomes using windows that contained at least 5 CpG (CHG or CHH) sites with a 2-fold change in methylation level. Two nearby DMRs would be considered interdependent and joined into one continuous DMR if the genomic region from the start of an upstream DMR to the end of a downstream DMR also had 2-fold methylation level differences between case and control. Otherwise, the two DMRs were viewed as independent. After iteratively merging interdependent DMRs, the final dataset of tDMRs was made up of those that were independent from each other.
Methylation of the target region
The target region was selected to design primers. First, net-PCR was used to amplify the DNA of the target region. Then the Illumina HiSeq 200 was used to sequence the designed DNA fragments. After calling methylation, data with bisulfite conversion rate < 98% were excluded. After preliminary analysis, data with an average CpG island coverage less than 20x and a loss rate greater than 0.20 were removed. Finally, samples with a missed detection rate greater than 0.30 were removed.
Articular processes were obtained from CS and control patients. Articular processes were fixed in formalin, decalcified, and embedded in paraffin. After sectioning, rehydration was performed. The sections were rinsed in xylene twice for 15 min each, rinsed in gradient ethanol solutions once (100%, 95%, 85%, and 70% for 5 min each), rinsed in double-distilled water (ddH2O) three times for 3 min each, and then treated with 3% hydrogen peroxide for 5 min. The sections were incubated with 3% bovine serum albumin for 30 min at room temperature and then incubated with the primary antibody (TNS3) at 37°C overnight. The sections were then incubated with a biotinylated goat anti-rabbit antibody for 30 min. Afterwards, the sections were incubated for 50 min with horseradish peroxidase-labeled streptavidin. DAB chromogen was added to develop the color for 30 s. Last, the sections were counterstained with hematoxylin for 30 s, and the slide was mounted for observation.
Results were recorded and analyzed by SPSS software (version 24.0; SPSS, Inc., Chicago, IL, USA). Clinical and biological data were assessed by independent t-test and Chi-square. Quantitative data are expressed as the mean ± standard deviation and were assessed by independent t-test. And numeration data were assessed by Chi-square. In the first stage, the DNA methylation difference between case and control group were compared using Fisher test; In the second stage, the DNA methylation difference between case and control group were compared using the U-test. The difference was considered significant if the p value was <0.05.