ONFH is a disease characterized by necrosis of one or more parts of the femoral head. ONFH can cause severe joint pain and limited physical activity, and some severe cases even require surgical intervention to restore normal function (Werner et al. 2003). Steroids are widely used in clinical practice. However, in recent years, steroids have become the leading cause of non-traumatic ONFH (Kerachian et al. 2009). We hope to find the genetic and epigenetic links between several important genes related to SONFH through the study of gene polymorphisms and DNA methylation.
In our case-to-case study, we did not find a relationship between rs10052957 and rs41423247 in NR3C1 gene and SONFH risk. However, in the further study of DNA methylation, compared with the control group, we screened for the first time the CpG sites of NR3C1_2-163 and NR3C1_4–47 with significant differences in methylation levels, and found that there was a correlation between NR3C1 gene SNPs and DNA methylation. According to previous researches, glucocorticoid is an important regulator of energy homeostasis. Most effects of glucocorticoids are mediated by the glucocorticoid receptor (Nicolaides et al. 2010). Glucocorticoid receptor is composed of DNA-binding domain, hormone binding domain and amino terminal region, and plays a decisive role in glucocorticoid sensitivity (Bamberger et al. 1995). Any of these changes in the molecular mechanisms of glucocorticoid receptor action can alter tissue sensitivity to glucocorticoid, possibly leading to drug resistance or allergy, and serious complications. NR3C1 polymorphisms are associated with changes in glucocorticoid sensitivity, body composition, and metabolic parameters. The polymorphism of rs41423247 is the change of C → G nucleotide downstream of the connection region of intron 2 of exon 2 of glucocorticoid receptor gene (van Rossum and Lamberts 2004). Rs41423247 polymorphism is associated with changes in susceptibility to exogenous glucocorticoid in rheumatoid arthritis and healthy individuals (van Rossum et al. 2003; Derijk et al. 2001). Yan et al. (2013) reported that in rs10052957, A allele carriers had significantly lower systolic blood pressure, total cholesterol and Low-density lipoprotein than GG carriers. People with GG, a homozygote of rs41423247, had a lower body mass index (BMI) and a lower triglyceride than those with the C allele. Although no polymorphism of NR3C1 has been reported on SONFH, the lower risk of blood lipids, systolic blood pressure, and other risk factors for the disease could be studied. In this study, we did not find the relationship between the SNPs allele frequency and genotype frequency of NR3C1 gene and the susceptibility to SONFH, which may be related to the study subjects we selected. The control group we recruited was from the same hospital, but the blood samples of SONFH patients were obtained from different regions and hospitals. It is well known that population confounding is a confounding factor in association analysis, which affects the results of association analysis. Further research is needed to control the interference of regional differences on the research results. Interestingly, this study showed that epigenetic modification of NR3C1 gene can interact with gene polymorphism, providing a new perspective for the pathogenesis of SONFH.
MTHFR is a key enzyme in homocysteine metabolism and folic acid metabolism. It can catalyze 5, 10⁃Methylenetetrahydrofolate to methylate homocysteine in blood and maintain its level in blood. The rs1801133 polymorphism of the MTHFR gene is caused by a point mutation at position 677, resulting in a decrease in enzyme activity that can cause hyperhomocysteinemia (Zhu et al. 2015). High concentration of homocysteine in the blood will interfere with the function of vascular endothelial cells, resulting in abnormal coagulation and fibrinolysis systems, making the body in a pre-thrombotic state. It can also act on endothelial cells to constrict blood vessels, increase blood pressure, and promote the formation of atherosclerosis. The growth of new blood vessels is the basis of blood supply, and angiogenesis is crucial for wound healing and bone remodeling in most tissues. Therefore, this study included MTHFR as a candidate gene. A genetic link between ONFH and a tendency to low fibrinolysis or thrombosis has been reported (Gagala et al. 2013). Clotting factors such as MTHFR and plasma plasminogen activation inhibitor − 1(PAI-1) are related to the occurrence of ONFH. Therefore, many studies have proposed that pathogenesis such as coagulation and angiogenesis may also be major risk factors for ONFH. Several case-control studies examined the relationship between the rs1801133 polymorphism of MTHFR gene and ONFH. However, there are inconsistent views on the role of this polymorphisms in the pathogenesis of ONFH, which is also an important factor for us to include rs1801133 in the study. Zalavras et al. (2002) reported a significant statistical difference between the incidence of non-traumatic femoral head necrosis and rs1801133 gene mutation. But there are also some research does not support this result, a south Korean contains 443 ONFH patients and 273 healthy subjects case-control study was carried out on 15 SNPs genetic analysis, found that rs1801133 polymorphism of MTHFR gene and non-traumatic ONFH and no obvious relationship between genetic susceptibility, the author thinks that different regions and ethnic may affect the MTHFR gene polymorphisms between individuals (Kim et al. 2010). Through further research, Shang et al. (2012) conducted a meta-analysis of the association between rs1801133 polymorphism and ONFH, and found in the Asian population cannot find rs1801133 polymorphism and the correlation of ONFH, but can be found in Africa, the crowd and the relative importance of ONFH, suggesting that rs1801133 polymorphism of MTHFR gene may not be a major risk factor for Asian populations ONFH, but a major risk factor for African people ONFH. When ethnicity was stratified, this meta-analysis provided no evidence of an association between rs1801133 polymorphism and ONFH. Twelve studies were included in the meta-analysis of Chai et al. (2015). The results showed that there was no direct association between ONFH susceptibility and rs1801133 polymorphism. Based on previous studies, we independently analyzed SONFH, hoping to explain the various associations between the MTHFR gene and steroid-induced ONFH reported in the literature. However, our results are also consistent with the results of most studies. We did not find a significant correlation between rs1801133 polymorphism of MTHFR gene and SONFH, and rs1801133 polymorphism may not be an explicit risk factor for thrombosis. Our study did not include an assessment of the mechanisms of hemostasis and, in addition, we did not measure homocysteine levels. The hypothesis that rs1801133 polymorphism may cause SONFH by promoting intravascular coagulation has yet to be confirmed. In addition, in order to study the interaction between SNPs and the methylation level at the site, the methylation level on the CpG island of the MTHFR gene amplicon was detected, and the methylation levels at the site, fragment and gene were statistically analyzed. The results showed that there were statistically significant differences in methylation levels at the sites of MTHFR_1–36, MTHFR_1–77, MTHFR_1-139 and MTHFR_2–42 compared with the control group. In the development of SONFH, epigenetics may be an important bridge connecting the interaction between environmental factors and genes.
Meanwhile, in the current case-control study, we investigated the relationship between rs2453839, rs3110697 of IGFBP3 gene and the risk of ONFH. We confirmed that rs3110697 polymorphism of IGFBP3 gene is related to the occurrence of SONFH in Chinese Han population. Compared with the control group, rs3110697 carriers with A/G genotype had A reduced risk of disease. In the recessive genetic model, the risk of rs3110697 A allele carriers was also reduced. We found that CpG locus IGFBP3_2-143 may interact with IGFBP3 gene polymorphisms, thus affecting the occurrence of SONFH.
IGFBP3 is the main carrier of IGF-1 in blood circulation and can prolong the half-life of IGF-1 in blood circulation. IGF-1 can promote the proliferation of bone cells, increase bone length, participate in the regulation of protein metabolism, induce vitamin D activation (Duchén et al. 2017). IGFBP3 plays a role in hypoxia-induced angiogenesis (Granata et al. 2007). Among IGFBP members, IGFBP3 is the most abundant in the circulatory system and is associated with arteriosclerosis (Jones and Clemmons 1995). Therefore, we believe that IGFBP3 gene polymorphisms may affect blood circulation, damage the femoral head, and further lead to SONFH. Multiple polymorphisms in the IGFBP3 gene are also associated with cancer risk. Previous studies have found a negative association between IGFBP3 rs2453839 and endometrial cancer risk, and a genetic variant of IGFBP3 may influence endometrial cancer risk in Caucasians (Mcgrath et al. 2001). Two IGFBP3 polymorphisms (rs3110697 and rs2854744) were significantly associated with premenopausal women with breast cancer survival (Deming et al. 2007). The relative specificity of IGFBP3 to ONFH has also aroused widespread concern. Jung et al. (2010) used Affymetrix targeted genotyping 3K microarray array to study the SNPs of IGFBP3 gene in 460 patients with ONFH and 300 control subjects. Rs2453839 was significantly associated with alcohol-induced and idiopathic ONFH in a recessive model. Subjects carrying the small homozygous allele CC of rs2453839 had a higher risk of ONFH. Song et al. (2012) reported that IGFBP3 rs2453839 TT and CT frequency of ONFH group increased and decreased compared with normal control group. The association analysis of polymorphism and clinical phenotype showed that the course of disease in patients with IGFBP3 gene rs2453839 TT ONFH was significantly shorter than that in patients with CT + CC. The frequency of CT + CC genotype in stage III / IV bilateral hip lesions group was significantly higher than that in stage III / Iv unilateral hip lesions group and stage II / III bilateral hip lesions group. However, we did not find a correlation between rs2453839 polymorphism and SONFH in this statistical analysis. The major models of IGFBP3 gene rs3110697 and rs2453839 were significantly associated with an increased risk of femoral head failure in 182 patients and 179 healthy controls. The genotype of rs2453839 was also associated with clinical stage. Compared with control group, IGF1 in ONFH group increased. Serum triglyceride and Low-density Lipoprotein levels in the ONFH group were significantly higher than those in the control group, but serum high-density Lipoprotein cholesterol levels were significantly lower than those in the control group (Song et al. 2016).
To sum up, our study proved for the first time that epigenetic modifications of NR3C1, MTHFR and IGFBP3 genes can interact with genetic polymorphism to influence SONFH in Chinese Han population. Based on the study of genetic structural characteristics of the disease, we can accurately grasp the development law of the disease. While there are some important findings in our study, there are some limitations. First, our study did not include an analysis of biological function, which is essential to elucidate the role of NR3C1, MTHFR, and IGFBP3 genes in SONFH. Secondly, SONFH needs to be further analyzed by different clinical stages. Thirdly, the control group in our study were all Han recruited from the affiliated hospital of neurology institute of Anhui university of traditional Chinese medicine, which may be a selection bias. Further functional studies and larger case-control studies are expected to avoid these issues and strengthen our conclusions.