Association of TIMP4 gene variants with steroid-induced osteonecrosis of the femoral head in the population of northern China

Introduction

Osteonecrosis of the femoral head (ONFH) is a common disease in bone and joint surgery, and is usually clinically classified as traumatic ONFH and atraumatic ONFH. The occurrence of ONFH is closely associated with several factors, including transcervical fracture, hormone intake, chronic alcoholism, sickle cell disease, and decompression sickness (Shibahara et al., 2000). From the initial hip osteoarthritis, followed by the eventual collapse and deformation of the femoral head, and then the eventual final hip dysfunction, ONFH has a high disability rate if not treated early. There are many effective therapies for ONFH, such as drug intervention and hip replacement therapy, and the number of patients with ONFH who seek treatment is gradually increasing. In 2004, a Japanese survey reported that nearly 11,400 ONFH patients require treatment yearly, with about 2,200 new cases reported each year (Kubo et al., 2016). In China, there are about 7 million patients with ONFH, and more than 100,000–200,000 new cases annually (Yin et al., 2014). In 1953, Pietrogrande and Marino first reported association of the use of glucocorticoids with ONFH (Pietrogrande & Marino, 1953). The treatment of some diseases such as asthma, rheumatoid arthritis, and lymphoid malignancies are dependent on the use of steroid hormones, and steroid intake was identified as the important cause of ONFH.

The development and progression of steroid-induced ONFH is a complex dynamic process mediated by multiple factors and signaling pathways, and is regulated by different genes. The disruption of the blood supply of the femoral head causes bone cell death, bone marrow edema, and eventually bone destruction and the collapse of the femoral head. Possible mechanisms that stimulate this process include high blood coagulation, thrombosis in blood vessels, and arterial contraction of the blood supply of the femoral head (Janke et al., 2013). Jaffe et al. first described the effects of corticosteroids to increase fat deposition in the intramedullary tissue of the femoral head, which can induce hyperlipidemia, resulting in increased cortical pressure, blood flow restriction, and local ischemia (Jaffe et al., 1972). With decreased number and activity of osteoblasts, apoptosis may also contribute to the pathological process of ONFH (Zhu et al., 2011). In recent years, single-nucleotide polymorphisms of some genes have been linked to steroid-induced ONFH. Research has been confirmed that variants of endothelial NO synthase (eNOS), Sex determining region Y-box 9 (SOX9),paraoxonase-1 (PON-1), Osteoprotegerin (OPG), and receptor activator of nuclear factor-kappa B ligand (RANKL) genes were closely correlated with the development of ONFH (Song & Du, 2016; Gagala et al., 2013; Wang et al., 2013; Li et al., 2016).

Matrix metall oproteinases (MMPs) and tissue inhibitors of metalloproteases (TIMPs) family are   widely   present in the extracellular matrix (ECM) of all tissues of our body, and also participate in the pathological processes of diseases. TIMPs can regulate proteolysis of the extracellular matrix by inhibiting MMP-dependent ECM proteolysis (Arpino, Brock & Gill, 2015). The extracellular matrix is an important extracellular component that provides structural and biological support for the surrounding cells, and its components are involved in cell adhesion, cell spreading, and cortical actin cytoskeleton organization (Gao et al., 2014). Guo et al. (2004) confirmed that the over expression of TIMP4 inhibited the formation of neointima of the carotid artery, inhibited the migration of vascular smooth muscle cells, and induced apoptosis. TIMP4 can contribute to hemostasis of the extracellular matrix in a tissue-specific manner (Gomez et al., 1997). The balance between MMP and TIMP plays an important role in regulating the balance of cartilage production and degradation. Expression of TIMP1 and TIMP4 was decreased significantly in osteoarthritis patients, which resulted in increased metalloproteinase, leading to pathological cartilage destruction (Kevorkian et al., 2004). Grässel, et al. confirmed that gene expression rates of MMP-2 and TIMP-1 were higher in bone tissue of ONFH, and the increase of MMP-2 biosynthesis and activation may counter-acting the production of new bone matrix (Grassel et al., 2010). In a study of a population of northern China, Jieli, Du. et al. indicated that MMP8 polymorphisms are significantly associated with susceptibility to steroid-induced ONFH (Du et al., 2016).

In the pathogenesis of steroid-induced ONFH, different signaling pathways and transcription factors play a key role. However, it is not clear whether mutation of the TIMP4 gene is involved in the development of steroid-induced ONFH. In this work, we analyzed SNP alleles, genotypes, and haplotypes in 309 patients and 286 controls to explore the potential association between TIMP4 single-nucleotide polymorphisms and steroid-induced ONFH in the population of northern China.

Materials and Methods

Results

Risk assessment between the TIMP4 haplotype and steroid-induced ONFH

In haplotype model analysis, one linkage disequilibrium (LD) block was detected in the TIMP4 SNPs (rs99365, rs17035945, rs308952, rs3817004, rs28897670 and rs227950; Fig. 1). Compared with the “CCGAAA” wild-type, the “TCAGAC” sequence was associated with a decreased risk of steroid-induced ONFH (OR = 0.71, 95% CI [0.53–0.95], p = 0.021; adjusted OR = 0.73, 95% CI [0.54–0.99], p = 0.04), and the “CCGGAA” sequence was also found to be associated with decreased risk after adjustment (OR = 0.31, 95% CI [0.10–0.98], p = 0.046) (Table 5).

Table 5:

The haplotype frequencies of TIMP4 polymorphisms and their association with steroid-induced ONFH risk in case and control subjects.

Compared with the “CCGAAA” wild-type, the “TCAGAC” sequence was associated with a decreased risk of steroid-induced ONFH (OR = 0.71, 95% CI [0.53–0.95], p = 0.021; adjusted OR = 0.73, 95% CI [0.54–0.99], p = 0.04), and the “CCGGAA” sequence was also found to be associated with decreased risk after adjustment (OR = 0.31, 95% CI [0.10–0.98], P = 0.046).

	Haplotype						Freq		Without adjustment		With adjustment
	rs99365	rs17035945	rs308952	rs3817004	rs28897670	rs227950	Case	Control	OR (95% CI)	p-value	OR (95%CI)	p-value
1	C	C	G	A	A	A	0.625	0.557	1.00	—	1.00	—
2	T	C	A	G	A	C	0.208	0.254	0.71(0.53–0.95)	0.021	0.73(0.54–0.99)	0.04
3	C	T	G	A	G	A	0.090	0.105	0.72(0.48–1.09)	0.12	0.76(0.50–1.16)	0.21
4	C	T	G	A	A	A	0.051	0.039	1.12(0.64–1.96)	0.68	1.23(0.68–2.21)	0.49
5	C	C	G	G	A	A	0.009	0.020	0.34(0.11–1.05)	0.062	0.31(0.10–0.98)	0.046

DOI: 10.7717/peerj.6270/table-5

Discussion

In clinical orthopedic practice, ONFH is a refractory disease, and about 80% of untreated patients suffer from destructive femoral head collapse (Mont et al., 2006; Mont, Jones & Hungerford, 2006). Determining the molecular basis of pathogenesis has gradually become the focus of research on the prevention and treatment of ONFH. Human genetic polymorphisms affect the susceptibility and tolerance of the human body to disease, clinical phenotypic diversity, and response to drug treatment. There is a potential association between multiple genetic polymorphisms and susceptibility to ONFH, including polymorphisms in the PPAR γ, RUNX2, COL2A1, IGFBP3, MMP2, and MMP8 genes (Du et al., 2016; Song et al., 2017; Yu et al., 2016). As an endogenous inhibitor of MMPs, TIMP4 can effectively inhibit the expression of MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9 (Liu et al., 1997), and gene variants may affect the risk of steroid induced ONFH. Through this case-control study, we explored the association between seven SNPs in TIMP4 and the risk of steroid-induced ONFH in the population of northern China . Based on our experimental results, SNPs rs99365, rs308952, rs3817004, rs2279750 and rs3755724 showed significant association with decreased susceptibility of steroid-induced ONFH. In addition, “TCAGAC” and “CCGGAA” sequences (rs99365, rs17035945, rs308952, rs3817004, rs28897670, and rs227950) were associated with 0.73-fold and 0.31-fold decreased steroid-induced ONFH risk respectively.

The appropriate assembly of the extracellular matrix (ECM) provides a suitable environment for the basic functions of cells, and changes of the ECM composition will affect embryonic development, morphogenesis, tissue remodeling, and repair (Brew & Nagase, 2010). There are four members of the TIMP family (TIMP1 to 4), which are involved in the degradation and composition of ECM (Bokarewa, Dahlberg & Tarkowski, 2005). The composition of ECM is dynamic, and this composition plays an important role in cartilage dynamic balance and cartilage repair (Ravindran & George, 2014; Yan & Zhang, 2006). The expression of TIMP4 also affects the damage and remodeling of cartilage and bone tissue. The expression of TIMP4 was markedly decreased in the cartilage of osteoarthritis patients (Kevorkian et al., 2004). However, according to an osteoarthritis study by Huang, TIMP-4 gene expression is increased in the cartilage of the human femoral head (Huang et al., 2002). In addition, Kumarasinghe et al. (2010) reported that that TIMP4 was significantly down-regulated in the bone of primary hip osteoarthritis, consistent with a role as a pivotal regulator of osteoblast expression. The necrosis of cartilage and subchondral bone eventually leads to the collapse of the femoral head, which is the main pathological change of ONFH (Song & Du, 2016). In ONFH, members of the MMPs/TIMPs family also participate in the accumulation and degradation of ECM in the cartilage and in the subchondral bone of the femoral head. In the extracellular matrix of cartilage, certain MMPs participate in the degradation of the ECM, and increased secretion of TIMP4 inhibits MMPs activity, to maintain cartilage self-renewal (Feng et al., 2013). Here, we report for the first time that five SNPs in TIMP4 are significantly associated with reduced risk of steroid-induced ONFH in the population of northern China. We speculate that TIMP4 likely involved in the pathological process of steroid-induced ONFH by regulating the extracellular matrix of the femoral head cartilage and subchondral bone.

Of the five SNPs in TIMP4 that showed significant association with decreased risk of steroid-induced ONFH, rs3755724 is located in the promoter region. Our results showed that allele C of rs3755724 was associated with a 0.78-fold decreased steroid-induced ONFH risk (OR = 0.78, 95% CI [0.621–0.985], p = 0.04). In the genotypic model analysis, rs3755724 was correlated with a decreased risk of steroid-induced ONFH in the Dominant model before adjustment. In a study of Korean children, an association of rs3755724 of TIMP4 with the development of Kawasaki disease with coronary artery lesions was identified (Ban et al., 2009). A study of cardiovascular disease showed that TIMP4 was up regulated in inflammatory processes of human cardiovascular pathology (Koskivirta et al., 2006). As an inhibitor of MMPs, TIMP4 is associated with smooth muscle cell migration and collagen deposition in vascular injury response (Dollery et al., 1999). Saito et al. studied the vasculitis caused by hormones and suggested that arteritis of the bone is a primary factor or a prerequisite for steroid-induced ONFH (Saito, Ohzono & Ono, 1992). TIMP4 is considered the main MMP inhibitor in human platelets, is involved in the regulation of platelet recruitment and aggregation, and its imbalance with MMP2 may be related to antithrombotic features (Radomski et al., 2002). In vulnerable subchondral capillaries and sinusoids, fibrin platelet thrombosis may be the main intermediate pathway of osteonecrosis of the femoral head (Jones Jr, 1993). Aller et al. (2017) reported the association of TIMP4 rs3755724 with predictors of ≥5% weight loss. Furthermore, in a study of obesity, TIMP4 was associated with high fat diet-induced obesity and may act through the regulation of lipid absorption (Sakamuri et al., 2017). There is a growing awareness that steroid hormones may cause ONFH through disruptions to lipid metabolism. Long-term high doses of steroid hormones intake induced hyperlipidemia and also caused hypertrophy and proliferation of fat cells in the bone marrow cavity of the femoral head, resulting in ONFH (Ding & Zhou, 2009). Therefore, we can speculate that TIMP4 may be involved in vasculitis formation, intravascular thrombosis and lipid metabolism disorders in hormone-mediated osteonecrosis of the femoral head.

Conclusion

Our case-control study is the first demonstration that five TIMP4 SNPs (rs99365, rs308952, rs3817004, rs2279750, and rs3755724) are significantly associated with decreased risk of steroid-induced ONFH in the population of northern China. The identification of these alleles is an important step towards screening gene variants for the prediction of disease and early clinical prevention. However, the pathogenesis mechanisms of steroid-induced ONFH are very complex, and to clearly understand the exact mechanisms of steroid-induced ONFH and the function of TIMP4 in the associated pathological changes, more samples from different regions for more detail and in-depth studies are needed.

Supplemental Information