Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Poznan University of Medical Sciences (protocol code Nos. 593/19 and 594/19, date of approval 6/19/2019). Informed Consent Statement: Written informed consent was obtained from all subjects or their legal guardians involved in the study.
The research involved a study group (N = 63) comprising 56 female and 7 male participants diagnosed with idiopathic scoliosis. All of these patients underwent posterior spinal surgery to correct the IS condition. To be included in the study group, all patients had to meet specific criteria: (1) a confirmed diagnosis of IS (other types of scoliosis were excluded); (2) the absence of any coexisting genetic, neurological, or orthopedic conditions; (3) the main curvature had to be located in the thoracic region; and (4) they had to undergo surgical treatment using posterior spinal instrumentation and fusion. Before the surgery, all patients underwent clinical and radiological assessments, which included long-cassette standing X-rays. The measurements taken included the number, location, and size of the curvature (Cobb angle). The patient's skeletal maturity was evaluated using the Risser sign. A spine surgeon performed all the measurements. Based on the final severity of the disease at skeletal maturity, the patients were divided into two subgroups: those with scoliosis equal to or less than 75° according to the Cobb angle (first subgroup), which consisted of 36 patients with a minor risk of significant impact on cardio-pulmonary function in adulthood, and those with scoliosis greater than 75° (second subgroup), which comprised 27 patients with severely progressive IS that could potentially affect cardio-pulmonary function.
A control group of (N = 30) 27 female and 3 male patients were included in the study, consisting of 23 healthy females, 2 patients with spondylolisthesis, 3 patients with scoliosis from prior thoracotomy, and 2 patients with Scheuermann's. The inclusion criteria for the control group were as follows: (1) an angle of trunk rotation of less than 3° at the examination with a scoliometer or clinically and radiologically confirmed diagnosis of spondylolisthesis, scoliosis secondary to prior thoracic surgery or Scheuermann's disease, (2) no coexisting orthopedic, genetic, or neurological disorders, (3) no diagnosis of IS, (4) surgical treatment due to spine pathology (5) no family history of idiopathic scoliosis. Clinical and molecular examinations were conducted to evaluate the patients from the study and control groups.
Genomic DNA was isolated from peripheral blood (collected in EDTA-containing tubes; Sarstedt, Germany) using the Quick-DNA Miniprep Kit (Zymo Research, Irvine, CA, USA) following the protocol provided by the manufacturer. DNA quantity and purity were determined using a spectrophotometer (NanoPhotometer NP80, Implen, Munich, Germany), which measured the absorbance at A = 260 and A = 230 nm, as well as the ratios A = 260/230 and A = 260/280. All samples analyzed met the purity criteria, with both ratios within the range of 1.9 to 2.0. A standard 1% agarose gel (LabEmpire, Rzeszow, Poland) electrophoresis was performed in the presence of ethidium bromide (50 ng/ml, Merck) to assess DNA integrity.
Total genomic DNA was processed as described before. After bisulfite conversion, the DNA was used as a polymerase chain reaction (PCR) template, followed by pyrosequencing (PSQ). The primers for both reactions were custom-designed using PyroMark Assay Design 2.0 software (version 2.0.1.15; Qiagen, Hilden, Germany) and then synthesized by Genomed (Warsaw, Poland). Two regions in the ESR1 and ESR2 genes were selected for analysis. The input DNA sequences corresponded to the T-DMR1, T-DMR2, and promoter 0 N and exon 0 N, respectively (https://www.ncbi.nlm.nih.gov; GenBank Nos.: NG_008493.2 and NG_011535.1; accessed on 5 March 2019). For the LBX1 gene, the analyzed DNA sequences were from both the forward and reverse strands of the promoter region (https://www.ncbi.nlm.nih.gov; GenBank No: NG_009236; accessed on 31 March 2018). Characteristics of the forward and reverse primers are shown in Table 1.
PCR reactions were conducted following the validated conditions for ZymoTaqTM PreMix (Zymo Research, Irvine, CA, USA). Details of the reaction components, concentrations, and thermal profiles are listed in Table 2.
The pyrosequencing analysis was carried out using the PyroMark Q48 instrument (Qiagen, Hilden, Germany). CpG assays were designed using Pyromark Q48 Autoprep 2. 4.2 software provided by Qiagen. For ESR1 T-DMR1 and T-DMR2 regions, we analyzed the methylation levels of 4 and 8 CpGs, respectively. ESR2 promoter 0 N and exon 0 N were analyzed for 16 and 19 CpGs. For each LBX1 strand, 15 CpG sites were analyzed within the promoter region.
Data analyses were performed using MedCalc 15.8 software (MedCalc Software Ltd, Ostend, Belgium). A Shapiro-Wilk test was used to examine the distribution of continuous variables. As the data were not normally distributed, nonparametric statistical tests were used to evaluate the differences in methylation levels between patients and controls, and the Mann-Whitney U test (adjusted for tied ranks) was applied. The Benjamini-Hochberg correction was used for multiple comparisons. The categorical data distribution was evaluated using Fischer's exact test.