Hypermethylation of the ALOX12 and CBS promoters in osteoporosis: Potential biomarkers for early diagnosis

Abstract

As a bone-confined chronic degenerative disorder, the progression of osteoporosis is characterized by abnormal crosstalk between osteoblasts and osteoclasts, leading to an imbalance of bone remodeling in adults. 1 Several studies reported that arachidonic acid ester 12 lipoxygenase (ALOX12) acts as a regulator in bone genesis by participating in the activation of the peroxisome proliferator-activated receptor γ (PPARG) pathway through its reaction product. 2 A positive correlation between ALOX12 gene polymorphism and bone mineral density (BMD) has also been verified, 3 indicating that serum Se deficiency was accompanied by some ALOX12 variation, contributing to the peak BMD and the development of osteoporosis. Another multidomain enzyme involved in DNA methylation, cystathionine beta-synthase (CBS), regulates the conversion of homocysteine into glutathione. Mutations in CBS lead to the production of more sulfur end-products from the methylation cycle. The newborn CBS-knockout (KO) mice who received treatment with recombinant poly ethylene glycol human truncated CBS (PEG-CBS) were rescued from osteoporosis-like symptoms. 4 In patients with osteoporosis, the expression of CBS was found to be down-regulated in femur tissues, leading to a lower BMD. 5 These findings support the idea that DNA methylation-related enzymes, including ALOX12 and CBS, are important for the occurrence of osteoporosis. However, the specificities in these 2 genes of osteoporosis human samples are largely unclear. Thus, we aimed to explore the impact of the DNA methylation level on select candidate genes in osteoporosis. These results will be of great help in optimizing treatments and for the early diagnosis of osteoporosis, complying with the principles of precision medicine. We first performed bisulfite conversion assays on both ALOX12 (Fig. 1A) and CBS (Fig. 1B). The sequencing results for the bisulfite-treated qMSP products are shown on the left part, confirming the complete bisulfite conversion. The right part shows the peaks after capillary electrophoresis, demonstrating that the lengths of the qMSP products were in line with the expectations. By characterizing the target sequence of human ALOX12 (Fig. S1B) and CBS genes (Fig. S1C), the methylation involving CpG islands in the promoter region on the qMSP primer fragments for both genes is highlighted in gray. Figure 1 Hypermethylation within the ALOX12 and CBS promoters in a rat model of osteoporosis and human osteoporosis patients. (A, B) qMSP results showing the methylation of ALOX12 (A) and CBS (B). (C) Representative micro-CT images of rats from the sham and ovariectomy groups. (D) Bone mineral density (BMD) analysis of rats in the two groups. (E) DNA methylation results for Alox12 and Cbs in the peripheral blood 12 weeks after surgery. (F) Western blot analysis of the ALOX12 and CBS expression in the rats. (G, H) Gray value statistical analysis of the CBS (G) and ALOX12 (H) expression. (I, J) Histograms showing the DNA methylation level of ALOX12 (I) and CBS (J). WB, whole blood. BM, bone marrow. (K, L) Correlation chart between age and DNA methylation level in all bone marrow osteoporosis samples. Fig. 1 To investigate the contribution of DNA methylation in ALOX12 and CBS during osteoporosis, we performed ovariectomy to induce osteoporosis-like symptoms in rats (Fig. S2A). Before we performed the surgeries, rats were pre-treated with antibiotics for 1 week and then observed for 12 weeks post-surgery (Fig. S2B). Micro-CT imaging and related analyses showed that the ovariectomized rats had a significantly decreased BMD compared with that in the sham group after 12 weeks (Fig. 1C, D). We further found a notable decrease in the bone volume fraction, trabecular thickness, and the trabecular number of the ovariectomized rats (Fig. S2C–E), demonstrating that our rat model had symptoms consistent with osteoporosis. We then studied the DNA methylation levels of ALOX12 and CBS in rats. After extracting DNA from whole blood samples of osteoporotic rats, we detected gene methylation based on PCR analyses. The results showed that the model group had higher methylation levels in both the ALOX12 and CBS genes compared with sham controls (Fig. 1E). Moreover, the protein expression of ALOX12 and CBS was also lower in the osteoporosis model group as indicated by Western blotting (Fig. 1F). The expression of ALOX12 was more extensively decreased (as indicated in the gray values) (Fig. 1G, H). Taken together, our experiments in the rat model demonstrate that there is hypermethylation of ALOX12 and CBS, and this correlated with the presence of osteoporosis and with decreased protein expression of these targets. To validate our above findings, a total of 46 osteoporosis patients with 30 age- and gender-matched normal controls were enrolled in this study as shown in Table 1. Whole blood and bone marrow samples were both collected for testing. We first performed a qMSP experiment to detect the methylation levels of ALOX12 and CBS in the patient samples. Our results showed that the ALOX12 gene methylation levels in the samples from patients with osteoporosis were significantly higher than those in normal controls (Fig. 1I). This was noted in both whole blood and bone marrow samples. In a further subgroup analysis, the bone marrow samples from males and whole blood samples from females with osteoporosis also had significantly higher ALOX12 methylation levels than the normal controls (Fig. S3A, B). However, there were no significant differences in the methylation levels of CBS between different genders or total samples (Fig. 1J; Fig. S3C, D), indicating the age impactor may play a more important role in integrating with CBS hypermethylation. Besides, the variations of ALOX12 and CBS in bone marrow samples with age showed clear positive correlations both in separate samples (Fig. 1K, L) and grouping analysis (Fig. S4A, C). Table 1 Baseline characteristics of the study participants. Table 1 Normal (n = 30) Osteoporosis (n = 46) P value Age (years) 58.33 ± 2.01 63.19 ± 2.03 0.123 Gender (male/female) 14 / 16 13 / 33 BMD (g/cm2) 1.01 ± 0.11 0.75 ± 0.10 <0.0001 WBCs (/μL) 5.00 (2.00, 21.50) 9.00 (4.50, 22.00) 0.178 RBCs (/μL) 13.00 (5.00, 20.00) 14.00 (7.00, 45.00) 0.219 EPIs (/μL) 4.00 (7.00, 15.00) 3.00 (6.00, 13.00) 0.325 Glucose (mmol/L) 5.15 ± 0.57 5.45 (4.90, 5.80) 0.073 UA (μmol/L) 299.15 ± 72.12 268.87 ± 84.60 0.080 Creatinine (μmol/L) 64.48 ± 12.32 61.71 ± 12.03 0.037 Urea (mmol/L) 5.09 ± 1.77 5.64 ± 1.65 0.127 T-BIL (μmol/L) 23.18 ± 8.69 21.25 ± 7.77 0.260 TP (g/L) 66.09 ± 4.47 65.78 ± 5.87 0.790 Albumin (g/L) 39.67 ± 3.37 37.03 ± 3.84 0.105 Ca (mmol/L) 2.19 ± 0.08 2.19 ± 0.10 0.907 CO2 (mmol/L) 26.00 (25.50, 27.00) 25.00 (25.00, 28.00) 0.096 Serum K (mmol/L) 3.77 ± 0.42 3.90 ± 0.48 0.164 Serum Na (mmol/L) 140.91 ± 2.04 142.00 (140.00, 143.00) 0.095 Serum Cl (mmol/L) 101.85 ± 1.91 103.00 (102.00, 104.00) 0.012 ALT (μ/L) 36.12 ± 3.22 40.26 ± 3.11 0.848 AST (μ/L) 27.30 ± 13.21 22.00 (19.00, 28.00) 0.926 γGT (μ/L) 26.68 ± 11.88 23.70 ± 9.84 0.196 ALP (μ/L) 68.38 ± 1.65 72.48 ± 2.01 0.157 LDH (μ/L) 499.00 ± 131.24 548.29 ± 112.29 0.053 An independent-samples T test was used. BMD: bone mineral density; WBC: white blood cell; RBC: red blood cell; EPI: epithelial cell; UA: uric acid; T-BIL: total bilirubin; TP: total protein; ALT: glutamic-pyruvic transaminase; AST: glutamic oxalacetic transaminase; γ-GT: γ-glutamyl transferase; ALP: alkaline phosphatase; LDH: lactate dehydrogenase. ∗Chi-square test was applied; aNonparametric test was used; bIndependent-Samples T test was used. Thus, we confirmed that there is hypermethylation of ALOX12 in both the rat model of osteoporosis and human patients with osteoporosis, suggesting that it may participate in the development of this disease. It may therefore have the potential as an easy-to-obtain early diagnostic biomarker for further investigation, especially for patients who cannot undergo x-rays or DEXA. It would also be a safe and cheaper alternative to DEXA which has therapeutic potential as well. Ethics declaration The human study was approved by the Ethics Committees of Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University (Shanghai, China) (No. 2021-YS-093). For age- and sex-matched rats, animals were fed and maintained under specific pathogen-free conditions following the criteria of the National Institutes of Health (Bethesda, MD) Guide for the Care and Use of Laboratory Animals with the approval of the Ethics Committees of Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University (Shanghai, China) (No. 2022–0271). Author contributions S.G. wrote the main manuscript and acquired the funding; Y.W. contributed to the methodology of experiments needed and data analysis; W.C. and P.H. did the investigation and data curation; W.C. reviewed the writing; H.W. did the validation; Y.W. and B.Z. made the supervision and reviewed and edited the manuscript. Conflict of interests The authors declare no conflict of interests. Funding This work is supported by grants from the Interdisciplinary Program of 10.13039/501100004921 Shanghai Jiao Tong University (No. YG2019QNA23).