Association of TNF-α-308(G/A) and -238(G/A) polymorphisms with non-traumatic osteonecrosis of the femoral head risks: a meta-analysis

Osteonecrosis is a potentially devastating condition in which the reduction of blood supply is the most significant motivator [1‐3]. When it comes to non-traumatic osteonecrosis of the femoral head (NONFH) which is a clinically common osteoarthropathy that mostly affects adults between 30 and 50 years old [4], the aetiology and pathology are not completely clarified [5]. Numerous risk factors have been identified that contribute to NONFH or the relevant pathological conditions, including hypercortisonism [6], hyperlipidemia, autoimmune diseases, dysbaric phenomena, smoking, hypofibrinolysis, alcoholism, and clotting disturbances [7, 8], which might result in intravascular coagulation and limited blood flow in caput femoris. Although plenty of risk factors have been recognized, the pathogenesis in molecular level remains unclear [9] and the therapy for osteonecrosis of femoral head continues to be a challenging.

Tumour necrosis factor (TNF), first reported in 1975 by Dr. Lloyd J. Old from Memorial Sloan-Kettering Cancer Center, New York, was described as a substance that mediates endotoxin-induced necrosis of tumors [10]. After years of investigation, TNF-α is generally known as a cell-signaling protein or cytokine participating in systemic inflammation and acute phase reaction. Produced mainly by macrophages as well as other immune effector cells and regulatory cells [11], TNF-α plays a rather intricate role in cellular processes: growth, proliferation, differentiation, and the immune reaction [12]. Meanwhile, the accommodative dysfunction of TNF-α has been reported to be implicated with many human disease such as inflammatory bowel disease (IBD) [13], chronic periodontitis [14], cancer [15, 16], psoriasis [17], several neurological disorders [18], et al. In relation to NONFH, elevated TNF-α serum concentration and expression of TNF-α in the bone marrow have been confirmed in both animal and clinical experiments during the process of steroid-induced osteonecrosis [19, 20]. The aggregation of macrophages into the necrotic area, resulting in the release of TNF-α together with other inflammatory factors, reinforces the inflammatory response and aggravates local osteoclasts [21].

TNF-α gene is located in the class III region of the major histocompatibility complex (MHC) and maps to 6p21.3 [22, 23]. It is revealed that gene polymorphisms in the regulatory region can generate the expression of TNF-α [22]. Recently, many genetic polymorphisms have been detected in the cytokine promoter at the initial position of TNF-α transcription site, which are 1031(T/C), 376(G/A), 308(G/A), 238(G/A), et al. [24]. Various scientific literatures have demonstrated TNF-α gene polymorphisms being associated to the susceptibility of systemic lupus erythematosus (SLE) [25], ankylosing spondylitis (AS) [26], gastric and hepatocellular carcinomas [27], and periodontitis [28], as well as NONFH [3, 29‐33]. Though many relevant research programs have been performed, when it comes to the relation of TNF-α-308(G/A) (rs1800629) and -238(G/A) (rs361525) polymorphisms to NONFH susceptibility, no common consensus has been reached yet and there does not exist any meta-analysis summarizing those relative studies. Therefore, we decided to perform a meta-analysis focusing on the association between TNF-α-308(G/A) (rs1800629) and -238(G/A) (rs361525) polymorphisms and susceptibility of NONFH to draw a precise evaluation of the relationship, which might be a potential treatment approach and could assist the early diagnosis of NONFH.

The risk factors for NONFH include internal and external elements, such as age, hypoxia, corticosteroid use, alcohol intake, smoking, and various chronic diseases (renal disease, haematological disease, inflammatory bowel disease, post-organ transplantation, and hypertension) [32, 40, 41]. Many factors were involved in the aetiology of NONFH, among which evidences supporting the TNF-α gene-308 or -238 polymorphisms have been provided currently [29‐33]. However, these distinct studies presented conclusions inconsistently. Therefore, we carried out this meta-analysis to reveal the association between TNF-α gene-308 or -238 polymorphisms and NONFH susceptibility.

Previous studies revealed that the TNF-α gene-308 polymorphism could be related to TNF-α gene regulation and associated with increased transcriptional activity of its product [32, 42, 43]. In our meta-analysis, overall samples revealed significant associations between TNF-α gene-308(G/A) polymorphism and NONFH susceptibility in alleles mode (G versus A) and recessive mode (GG versus GA/AA) (Table 3 and Fig. 2), which indicated that G allele may be a protective factor in the risk of NONFH. However, by comparing genotype GG with GA + AA, Biao-Fang Wei et al. found that the TNF-α mRNA and the TNF-α cytokine expression were not significantly different [29]. Thus, in-depth studies were required to identify the effects of TNF-α gene-308(G/A) polymorphisms on the development of NONFH. After analyzing by ethnicity, we found significant association in Asians but not in Caucasians, which suggested that the role TNF-α gene-308 polymorphism played in the risk of NONFH in Asian population was more notable than that in Caucasian population. The reasons why the SNP polymorphism in the same position of TNF-α gene played absolutely different roles in different races may lie on two aspects. First, the genetic backgrounds may differ from the two ethnic groups. Besides, there are numerous aetiological factors contributing to NONFH such as hyperlipidemia, autoimmune diseases, alcoholism, et al. [7, 8]. The varied prevalence of hyperlipidemia [44], autoimmune diseases [45], discrepant alcohol consumption [46, 47], et al., in different populations may explain the inconsistency. By the way, due to the fact that P for HWE was too small (Table 2) in study conducted by S. Samara et al. [32], we removed it in the sensitivity analysis. Consequently, homozygote mode (OR = 0.347, 95% CI 0.149–0.805, P = 0.014, Fig. 4b) and dominant mode (OR = 0.365, 95% CI 0.159–0.841, P = 0.018, Fig. 4c) showed different results, which indicated that the improper selection of the controls labilized the pooled estimate. Nevertheless, the deletion of Samara et al. [32] increased the number of mode type that revealed significant association.

Another common SNP of TNF-α we paid attention to is TNF-α gene-238G/A polymorphism. Samara et al. reported a G-to-A change at position 238 resulted in an up-regulation of TNF-α gene expression and suggested that the increase of TNF-α expression could lead to osteoclasts proliferation, which contributed to NONFH [32]. On the contrary, another study recently showed that the A-238 allele at position 238 may be associated with downregulation of tissue inflammation [43]. We conducted a meta-analysis including three eligible case-control studies containing 275 cases comparing 610 controls. The allele A was so rare that neither NONFH patients nor controls were found carrying AA genotype of TNF-α gene-238. For this reason, only the alleles mode (G vs. A) and recessive mode (GG vs. (GA + AA)) were conducted in the meta-analysis. The total sample demonstrated that statistical significance existed in both of the two models. The allele A at position 238 could be a risk factor to NONFH.

Tumour necrosis factor (TNF-α) gene is located on human chromosome 6p21.3 in the vicinity of the major histocompatibility complex (MHC) class III region [48] and plays an important role in inflammation, immunity, and cellular organization of its product. As we discussed above, the TNF-α gene-308 or -238 affected regulation of TNF-α gene and associated with altered transcriptional activity in many diseases. What is more, a number of studies had shown that the TNF-α promoter polymorphisms have a vital effect on transcriptional activity [49, 50]. It is generally believed that the pathophysiology of NONFH is related to the apoptosis of osteoblasts and osteocytes. Shibahara et al. suggested that there were mass apoptosis cells in necrotic zone and the apoptosis of osteocytes resulted in the osteonecrosis and the destruction of bone structure [51]. Besides, it is known that some cytokines such as IL, TNF-α, TNF-β, and other inflammatory-related cytokines play an important role in the balance between osteoclasts ad osteoblasts. Dai CY et al. have reported that TNF-α acts on osteoblasts or bone marrow cells to synthesize and release cytokines which is directly associated with osteoclasts proliferation and maturation [52]. Thus, we could assume that the TNF-α gene-308 or -238 polymorphisms led to an altered expression of the gene, which made a contribution to inflammation response. Consequently, apoptosis of osteoblasts and proliferation of osteoclasts were activated or aggravated, resulting in the deterioration of NONFH.

To the best of our knowledge, this is the first meta-analysis to comprehensively assess the association between TNF-α gene-308(G/A) and -238(G/A) and the susceptibility of NONFH, which may provide the clues of prevention and early diagnosis and potential trials for osteonecrosis treatment. During the searching process, we have followed specific and repeatable strategies and applied strict inclusion and exclusion criteria to recruit. We also considered the distribution of ethnic groups and applied subgroup analysis to assess the association in Asian and Caucasian populations, respectively. Furthermore, Clark scores was used to assess the quality of each studies and most of them possessed high qualities. HWE was used to test the homogeneity between controls and natural populations, and only one study was significantly deviated. Besides, no significant heterogeneity was identified among the enrolled studies. Despite of the insufficient research number, we still applied the Egger’s and Begg’s test and found no publication bias in the recruitment.

Nevertheless, a few limitations shall be claimed in this meta-analysis. First, the language was limited to English and Chinese, which may develop bias as a consequence of the restrictedly defined populations. Second, only two out of five studies mentioned the severity of NONFH. Thus, the limited number defies further analysis of the associations between TNF-α gene-308(G/A) and -238(G/A) polymorphisms and clinical stages or features of NONFH. Third, following the enrollment strategies, we excluded some researches due to lack of target SNPs data, which may increase the potential of selection bias. Fourth, in the sensitivity analysis, the ORs’ significance changed for TNF-α gene-308(G/A) polymorphism in homozygote mode and dominant mode after we removed S. Samara et al. [32]. As shown in Table 2, the genotyping variance of control group in S. Samara et al. [32] was derived from HWE, which might be the reason of the unstable results. Additionally, the removal of Yaosheng Liu et al. [30] also influenced the ORs’ significance for TNF-α gene-308(G/A) polymorphism in alleles mode and recessive mode. The weights of Yaosheng Liu et al. [30] in the meta-analysis of alleles mode and recessive mode were much heavy, which were 34.46 and 27.93%, respectively. Thus, the significant effects of this research indicate that further original studies are necessary to enhance the result stability.

This meta-analysis shows that TNF-α-308(G/A) and -238(G/A) polymorphisms are inversely associated with the risk of NONFH, while the significant association for 308(G/A) is observed in Asian. Original and intensive researches of individuals of diverse ethnicities would be essential due to the insufficient or conflict interpretations of the polymorphism effects on the NONFH pathology.