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

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. 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 proliferatoractivated receptor g (PPARG) pathway through its reaction product. A positive correlation between ALOX12 gene polymorphism and bone mineral density (BMD) has also been verified, 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. In patients with osteoporosis, the expression of CBS was found to be downregulated in femur tissues, leading to a lower BMD. 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

Hypermethylation of the ALOX12 and CBS promoters in osteoporosis: Potential biomarkers for early diagnosis 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 proliferatoractivated receptor g (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 downregulated 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.
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. S2CeE), 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.
Peer review under responsibility of Chongqing Medical University.
Available online at www.sciencedirect.com ScienceDirect journal homepa ge : www.keaipubl ishing.com/en/j ourna ls/genes-diseases 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).
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).

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.