DPP4 rs16822665 and rs2268694 had Protective Effect on Osteonecrosis of the Femoral Head

Background: It is reported that DPP4 is associated with bone metabolism, osteoporosis and other orthopedic diseases, but the correlation between DPP4 and osteonecrosis of the femoral head (ONFH) is not clear. It was the purpose of this study that was to explore the relationship between DPP4 gene and ONFH. Methods: We genotyped four single nucleotide polymorphisms (SNPs) from DPP4 gene using the Agena MassARRAY platform. The association between DPP4 variants and ONFH susceptibility was assessed using odds ratio (OR) and 95% condence intervals (CIs) via logistic regression. Results: The results showed that the allele C of rs16822665 was related to a lower risk of ONFH (OR = 0.76, 95%CI = 0.63-0.92, p = 0.006). In the case of stratied analysis, we found that rs16822665 could reduce the incidence of ONFH risk in four genetic models (dominant, codominant, log-additive, and recessive models) in drinkers and people age ≤ 51 years (p < 0.05). In gender stratication analysis, both rs2268694 and rs16822665 were contributed to bring down the risk of disease, which were mainly reected in the codominant, dominant and log-additive models in female (p < 0.05). The subgroup analysis was conducted based on smokers revealing that rs2268894 was vitally correlated with a decreased risk of ONFH in the codominant (C vs. T: OR = 0.51, 95% CI: 0.34-0.76, p = 0.001), dominant (TC-CC vs. TT: OR = 0.53, 95% CI: 0.36-0.77, p = 0.001), and log-additive (OR = 0.65, 95% CI: 0.48-0.88, p = 0.006) models, while it was not found in the non-smokers. Conclusions: This nding provide evidence that DPP4 variants play a key role in the occurrence of ONFH among the Chinese Han population. a major impact on glucose metabolism, immune regulation, signal transduction, cell migration differentiation 23 . Study have shown that the expression of CD26 mRNA in patients with rheumatoid arthritis is related to disease activity and bone erosion, suggesting that this molecule may play a role in the immunopathology of rheumatoid arthritis and bone erosion 21 . Some researches 24 found that higher plasma DPP4 levels were signicantly associated with higher bone turnover and a higher incidence of osteoporotic fracture. These results showed that DPP4 may be related with osteoporotic fracture by mediating bone turnover rate. Carbone et al found that in elderly group,there was no relationship between DPP4 and osteoporosis 25 . These lines of evidence have demonstrated that DPP4 gene played a crucial role in bone-related disease. However, the association between DPP4 gene and osteonecrosis susceptibility has not been reported. Rs2268694 and rs16822665 polymorphisms, located in the DPP4 gene protected susceptibility to ONFH was indicated in our results, but had not been previously reported in other diseases.


Introduction
Osteonecrosis of the femoral head (ONFH) is a common disease in orthopedics, which is di cult to treat 1  Femoral head necrosis has a high disability rate, which has a strong impact on the quality of life of patients. In recent years, some progresses have been performed in its treatment, but its speci c pathogenesis needs to be further studied.
The human DPP4/CD26 gene, located at 2q 24 , is a transmembrane glycoprotein with a molecular weight of 110ku and contains 26 exons encoding 766 amino peptides 6, 7 . Its product is a proteolytic enzyme, which has a unique proteolytic effect and belongs to the serine protease family 8, 9 . In addition, scholars found that DPP4/CD26 was involved in physiological processes such as invasion, apoptosis, migration, adhesion, and immune modulation 10 . As a marker of T-cell activation, DPP4/CD26 also plays a key role in immune regulation and activates broblast proteins and participate in signal pathways [11][12][13] .
It is well known that DPP4 and some of its substrates interact with adipokines, playing a pivotal role in the energy metabolism 14 . In recent years, DPP4 not only plays a vital role in protein regulation in the context of energy metabolism in the literature, but also has a direct and indirect effects on bone metabolism. Many substrates of DPP4 have played role in bone metabolism, and that include incretins, neuropeptides and gastrointestinal peptides. Overall, although some effects are conducive to bone formation, others are intricate and have not been absolutely expounded.
The activity of DPP4 is related to diabetes hyperglycemia, osteoporosis bone loss 15 and general osteoporosis, which indicates that the regulation of DPP4 is not directly involved in pathophysiology of these bone diseases, is also in uenced by its development.
Experimental studies have clearly con rmed some effects of DPP4 matrix on bone 16, 17 . In addition, some meta-analyses of clinical studies on DPP4 inhibitors and GLP-1-based bone therapy have been published 18 and have indicated that DPP4 inhibitors are relevant to a reduced fracture risk 18 . DPP4 inhibitor can improve vertebral bone mineral density and trabecular structure in vivo 19 , suggesting a harmful effect of DPP4 on bone metabolism. Studies have shown that DPP4 can digest several molecules playing an important role in bone metabolism. It was found that inhibition of DPP4 reduced serum reabsorption markers and trabecular bone volume and increased cortical and bone strength in rats with diabetes 20 . Yeganeh1 et al. 21 showed that DPP4 may be directly involved in bone resorption. In recent years, studies have displayed that the development of human osteoclasts is hindered when DPP4 signaling is blocked 22 .
So far, DPP4 has been reported to be associated with bone formation, bone loss, and osteoporosis. However, the mechanism of DPP4 gene effect on osteonecrosis remains unclear. In this study, we discussed the association of DPP4/CD26 genetic variations with ONFH susceptibility among Han individuals in China, which will help understand the function of DPP4/CD26 in the development of ONFH and provide new insights into its pathogenesis.

Study population
In this study, we enrolled 936 subjects, including 468 cases and 468 health controls. All cases were newly diagnosed by the following criteria: 1) diagnosed and selected by anteroposterior and bilateral hip X-ray lms and /or magnetic resonance imaging; 2) No other direct trauma, cardiovascular disease, rheumatoid arthritis, ankylosing spondylitis, hip joint disease (such as dysplasia of the hip), diabetes, renal insu ciency, cancer, glucocorticoid, alcohol and family genetic diseases were included. The selection criteria of all healthy people were: 1) they are from the same hospital during the same period; 2) People who are not long-term users of alcohol and steroids; 3) no pain in the buttocks; 4) no lesions were found in the pelvic anteroposterior lm and frog leg lateral lm.

DPP4 genotyping
Four SNPs (rs2268894, rs16822665, rs6741949, and rs67399148) in DPP4 gene were selected from 1000 Genomes Chinese Han Beijing population, with minor allele frequency (MAF) >5%. 5ml of fasting peripheral venous blood was collected from all subjects, and dispensed in ethylene diamine tetraacetic acid (EDTA)-containing tubes. Genomic DNA was puri ed with a commercially available DNA extraction kits (GoldMag Co. Ltd, Xi′an, China). Genotype analysis of DPP4 gene polymorphism was performed according to the protocol using Agena MassARRAY platform (Agena Bioscience, San Diego, CA, USA). The genotyping results were managed and analyzed using Agena Bioscience TYPER software (version 4.0).

Statistical analysis
Student's t-test was used to assess the difference of age, while χ 2 test was to compare the distributions of gender between the two groups, assess the frequency of SNP allele between the case and control groups, and gure up the Hardy-Weinberg equilibrium (HWE) of the control group. Odds ratio (OR) and 95% con dence intervals (CIs) were using for evaluating the relationship between DPP4 polymorphisms and ONFH risk by logistic regression analysis. Then, the interaction of SNP-SNP in the risk of ONFH was detected by multifactor dimensionality reduction (MDR).

Basic information of research objects
As rendered in Table 1

As shown in
After strati ed analysis (Table 4), we found that rs16822665 could reduce the incidence of ONFH risk in the four genetic models (dominant, codominant, log-additive, and recessive models) in drinkers and patients age ≤ 51 years (p < 0.05). In gender strati cation analysis, both rs2268694 and rs16822665 were contribute to bring down the risk of disease, which were mainly re ected in the dominant, codominant, and log-additive models in female (p < 0.05).
The subgroup analysis, which was conducted based on smokers revealed that rs2268894 was vitally related with a decreased risk of To sum up, we realized that two SNPs (rs2268894 and rs16822665) were not associated with patients at age > 51 years, non-smokers, and non-drinkers, suggesting that age, gender, smoking, and drinking are signi cant factors that affect the risk of ONFH.

SNP-SNP interactions
We analyzed the SNP-SNP interactions on the risk of ONFH by using the MDR. Table 5 represents that the best four-locus model including rs2268894, rs16822665, rs6741949, and rs67399148 was the best model to predict ONFH risk (training accuracy: 0.569; testing accuracy: 0.534; CVC = 10/10, p < 0.001).

Discussion
To the best of our knowledge that genetic studies have offered insights into a great number of diseases, including ONFH. In this study, it was expounded that DPP4 genetic polymorphisms were associated with ONFH risk among Han individuals in China. Allele, genotype of four loci between patients with ONFH and healthy samples were compared, and strati cation analysis were marched. We obtained that the allele C of rs16822665 was related to a decreased risk of ONFH in the Chinese Han individuals. In addition, the results showed that rs2268894 and rs13822665 have a protective effect on the risk of ONFH in patients age ≤ 51 years, or females, or smokers, or drinkers. Therefore, all of those date underscore the importance of DPP4 in ONFH development, and those two SNPs may become new biomarker for the early treatment and prevention of ONFH.
Dipeptide peptidase-4 (DPP4), also known as CD26, is an exopeptidase that is widely expressed on diverse types of cell surface 23 . By controlling the activity of its substrate or by physical binding with other proteins, the activity of this transmembrane exopeptidase has a major impact on glucose metabolism, immune regulation, signal transduction, cell migration differentiation 23 . Study have shown that the expression of CD26 mRNA in patients with rheumatoid arthritis is related to disease activity and bone erosion, suggesting that this molecule may play a role in the immunopathology of rheumatoid arthritis and bone erosion 21 . Some researches 24 found that higher plasma DPP4 levels were signi cantly associated with higher bone turnover and a higher incidence of osteoporotic fracture. These results showed that DPP4 may be related with osteoporotic fracture by mediating bone turnover rate. Carbone et al found that in elderly group,there was no relationship between DPP4 and osteoporosis 25 . These lines of evidence have demonstrated that DPP4 gene played a crucial role in bone-related disease. However, the association between DPP4 gene and osteonecrosis susceptibility has not been reported. Rs2268694 and rs16822665 polymorphisms, located in the DPP4 gene protected susceptibility to ONFH was indicated in our results, but had not been previously reported in other diseases.
Although our study found that the relevant loci of DPP4 gene have a protective effect on ONFH disease, which can offer a new theoretical basis for disease treatment and prevention, several potential limitations are unavoidable in our study. First, all participants were Han ethnicity, so we need more different ethnic populations to con rm our ndings. Second, the sample size is too small to con rm our results abundantly. Third, only four polymorphisms in DPP4 gene were studied, more polymorphisms are needed to be investigated.

Conclusions
All in all, we con rmed that DPP4 variants play a key role in the occurrence of ONFH among the Han population in China. This nding may provide new sights for the prevention and diagnosis of ONFH.

Declarations
Ethics approval and consent to participate The ethical approval of this study is in line with the ethical principles of the Helsinki declaration on human medical research. Our study has been approved by the ethics committee of Hong Hui hospital in Xi 'an, China, and all participants have signed informed consent before participating in the study.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

Funding
There was no funding support for this work.
Author's contributions Chang Liu designed this study protocol and supervised the study; Xuan Liu drafted the manuscript and performed the DNA extraction and genotyping; Xiaowei Li performed the data analysis and performed the sample collection and information recording. All authors have read and approved the nal manuscript.      Bold values indicate statistical signi cance (p < 0.05).