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Med One 2016;1(4):4;DOI:10.20900/mo.20160017
1Department of Orthopedics, Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine, Changsha, Hunan 410006, P.R. China;
2Department of Anatomy, Changsha Medical University, Changsha, Hunan 410219, P.R. China;
3Department of Clinical Laboratory, Changsha Stomatological Hospital, Changsha, Hunan 410004, P.R. China
First Author: Hui Xu, Associate Chief Physician, Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine. Email: firstname.lastname@example.org;
Correspondence: Jie Luo, Department of Clinical Laboratory, Changsha Stomatological Hospital, Hunan, P.R. China. Email: email@example.com.
Objective: To investigate the correlation between TGF-β1-509C/T gene polymorphisms and primary knee osteoarthritis (PKOA) to provide a new method for the pathogenesis, diagnosis, and treatment of PKOA.
Methods: Eighty eight PKOA patients and 89 cases of healthy control group were randomly selected from the Department of Orthopedics, the Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine. Polymerase chain reaction - restriction fragment length polymorphism (PCR - RFLP) analysis was performed to test the TGF-β1-509 locus genes and genotypes of the selected subjects. The genotypes and allele frequencies were compared with the chi-square test. The relative risk was represented with odds ratio (OR) and 95% confidence interval (CI).
Results: The distribution of TGF-β1-509 genotypes for PKOA and healthy control groups complied with Hardy-Weinberg equilibrium law, indicating a representative population was used in this study. Compared to the healthy control group, PKOA group had a distribution difference for both genotypes and allele frequencies. CC genotypic frequency of PKOA group was significantly higher than that of healthy control group (44.3 % vs. 13.5 %, χ2 = 20.51, P = 0.0000); allele frequency was significantly higher than that of control group (65.9 % vs. 41.6 %, χ2 = 21.08, P = 0.0000). The differences were statistically significant (P < 0.05). No statistically significant differences were found between mild and severe CC genotype frequencies (39.5 % vs. 48.9 %, χ2 = 0.792, P = 0.37), and between C allele frequencies (62.8 % vs. 68.9 %, χ2 = 0.728, P = 0.39) in PKOA group.
Conclusion: TGF-β1-509C/T gene polymorphism was associated with the PKOA in the patients in Hunan region. The allele C carriers in the locus might be more susceptible to the PKOA.
Primary knee osteoarthritis (PKOA) is a common and frequently occurred disease in the osteoarthropathic surgery and its incidence tends to grow constantly. It has become one of the chronic diseases that seriously harm the health of middle aged and elderly people . Scholars have widely and profoundly researched the etiology, mechanism, prevention and treatment of this disease. However, the pathogenic mechanism remains unclear and it cannot be easily found in an early period . The generation and development of PKOA is caused jointly by systematic factor, local factor, genetic factor, environment factor and other factors [3,4]. This study focuses on the correlation between TGF-β1-509C/T polymorphisms and PKOA in patients in Hunan region to open up a new field for the treatment of PKOA.
Eighty eight patients with PKOA (43 mild and 45 severe cases) who were diagnosed in the outpatients and inpatients of Department of Orthopedics in the Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine from May 2013 to September 2014 were selected randomly, without blood relationship among them, including 32 male patients and 56 female patients, aged 47.5 ± 8.3 years old. Inclusion criteria: 1) Patients whose clinical symptoms comply with the standards of the Guide of Chinese Orthopaedic Association of Chinese Medical Association on Diagnosis and Treatment of Osteoarthritis (2007). Osteoarthritis was divided into five levels according to Kellgren-Lawrence (K-L) X-ray grading criteria . According to clinical symptoms and in combination with knee x-ray K-L criteria, level 0, I or II belonged to mild PKOA; according to clinical symptoms and in combination with knee x-ray K- L criteria, level III or IV belonged to severe PKOA. 2) Patients without secondary knee osteoarthritis; 3) Patients without hypertension and diabetes and liver & kidney dysfunction. Eighty nine patients without PKOA clinical symptoms for physical examination and without significant abnormity for knee x-ray examination in the same period were selected randomly as the healthy control group, including 28 male patients and 61 female patients, aged 48.8 ± 7.5 years old.1.2 Reagents and Instruments
In this study, the following reagents and instrument were used, including Taq DNA polymerase 5 U/μL (Guangzhou Dongsheng Biotech Co., Ltd), restriction endonuclease Bsu 500 U/mL (MBI Fermenas), whole blood genomic extraction kit, DNAgel extraction kit (Guangzhou Dongsheng Biotech Co., Ltd), PCR instrument (ABI corporation), electrophoresis (Beijing Liuyi Biotechnology Co., Ltd), and integrated gel imaging analysis system (Beijing Sage Creation Science Co., Ltd).1.3 Gene Polymorphisms Testing
The fasting peripheral venous blood samples of 2 ml were taken from each subject in the early morning. The samples were subjected to an anticoagulation with EDTA-Na2 and then stored at 4 °С. Genomic DNA was extracted within one week. If it was not performed within one week, the samples should be stored at -20 °С. The genomic DNA extraction of peripheral bleed was performed with reference to the instructions of the kit. According to the references , the sequences of genes were found in NCBI database (rs1800469), and the PCR primer was designed with the software Primer 5.0 (sequence of forward primer: 5'-GCTACGGCGTGGAGTGCTGA-3'; sequence of reverse primer: 5'-AGAGGACCAGGCGGAGAAGG-3'). The primer sequences were synthesized by BGI Gene Research Center. The length of amplified fragment was 506 bp. PCR reaction conditions were pre-degenerated at 94 °С for 5 min, followed by 35 cycles at 94 °С, 30 sec., 56 °С 30 sec., and 72 °С 30 sec., and finally, 72 °С 5 min and stored at 4 °С. The amplified products were digested via endonuclease Bsu36I (Eco81L), and the digested products were subjected to the electrophoresis of 10g/L agarose gel.1.4 Statistical Analysis
The statistical analysis was performed with the statistical software SPSS13.0. The Hardy-Weinberg equilibrium, genotype and allele frequencies were compared with the chi-square test and the relative risk was represented with the odds ratio (OR)and 95% confidence interval (CI). P < 0.05 indicates the statistically significant difference.
If -509 locus (-CTTC-) existed in the amplified fragments, the interchange between thymine (T) and cytosine (C) might appear, namely, at -509 locus (-CTTC-), Bsu36l endonuclease locus would be generated, and it would produce two fragments: 329 bp and 177 bp after the digestion of Bsu36l endonuclease. TT geneotype had only one fragment (506 bp), CC genotype had two fragments (329 bp and 177 bp), and CT genotype had 3 fragments (506 bp, 329 bp and 177 bp) (Fig. 1).
Three genotypes were selected randomly and sequenced, respectively. The sequenced results were completely consistent with the electrophoresis results (Fig. 2).
Upon the tests, the distribution of TGF-β1-509 genotypes for both PKOA group and healthy control group complied with Hardy-Weinberg equilibrium law (χ2 = 0.135, P = 0.934; χ2 = 2.190, P = 0.335, P > 0.05), indicating a representative population was used in this study. Compared to the healthy control group, PKOA group had a distribution difference between genotype and allele frequency. CC genotypic frequency of PKOA group was significantly higher than that of healthy control group (44.3 % vs. 13.5 %, χ2 = 20.51, P = 0.0000); allele frequency was significantly higher than that of control group (65.9 % vs. 41.6 %, χ2 = 21.08, P = 0.0000) (P < 0.05). The analysis of relative risk of allele frequencies showed that the risk of C allele carriers to suffer from the PKOA was 2.72 times that of T allele (OR = 2.72, 95 % CI = 2.57 - 2.88, P = 0.0000) (Table 1).
Notes: # χ2=20.51, P=0.0000;*χ2=21.08, P=0.0000,OR = 2.72, 95%CI = 2.57-2.88, P =0.0000
No statistically differences were found between mild and severe CC genotype frequencies (39.5 % vs. 48.9 %, χ2 = 0.792, P = 0.37) in PKOA group and between C allele frequencies (62.8 % vs. 68.9 %, χ2 = 0.728, P = 0.39), the differences were not statistically significant, see Table 2.
OA incidence is a complicated pathological process. Currently, the basic researches of PKOA mainly focus on cartilage and synovium. The cytokines that can affect the cartilage cells and synovial cells mainly include growth factors, chemokines, interleukins, and tumor necrosis factors. Of them, TGF- β is considered to play an important role in the OA osteophytes and synovial hyperplasia. TGF- β1 belongs to the super-family of TGF- β, playing an important role in the growth and re-construction of cartilages and it is an important PKOA-associated gene. Many studies demonstrate the association between single nucleotide polymorphism (SNP) locus of TGF- β1 gene and the OA. However, the research results are different. Youcheng Wang  found that the TGF-β1 C1348-T locus polymorphism was significantly associated with the PKOA. The common variation in Chinese people occurs mainly at -509C/T, so the studies on the correlation between -509C/T locus and PKOA have not been reported at home and abroad.
The correlation analysis was performed in this study. The subjects in the analysis were the two populations selected from PKOA group and control groups, respectively, who have the same or at least very similar genetic background, with the purpose to compare the difference of allele frequencies of certain candidate gene polymorphism between the two populations. The correlation analysis shows that the distribution of the allele frequency in the PKOA group will be significantly higher than that in the control group if certain allele itself or its adjacent locus is associated with the disease susceptibility, and the allele frequency in the PKOA group will be significantly lower than that in the control group if it is related to the disease resistance and protection. The said experiments concluded that there was a difference of genotypic frequency and allele distribution between PKOA group and healthy control group. CC genotypic frequency in PKOA group was higher than that in healthy control group (44.3 % vs. 13.5 %), the C allele frequency was higher than that in healthy control group (65.9 % vs. 41.6 %), and the difference was statistically significant (P < 0.05). Logistic regression analysis result shows that, compared to the population carrying allele T, the risk of population carrying allele C to suffer from PKOA is 2.72 times (OR = 2.72, 95% CI = 2.57 - 2.88). This indicates that -509 allele C in TGF- β1 promoter region may be the susceptible gene of PKOA and the allele T may be the protective gene. This may be related to the reasons that the change of allele C toward allele T increased the translation and expression level of TGF- β1, inhibited the activation of immune cells and thus increased the resistance against the osteoarthritis , or related to the reasons that the high expression of TGF- β1 mediated the cartilage synthesis, inhibited the decomposition of collagen and proteoglycans, and protected the cartilage matrix from being hydrolyzed and damaged by proteases to achieve the damage reversal of cartilage [9,10].
Jing Guo, et al  found that the apoptosis of cartilage cells and synovial cells in OA group was negatively correlated with the expression of TGF-β1, the high expression of TGF-β1 in OA group might negatively regulate the generation of apoptosis of cartilage and synovial cells, and delay the generation and development of osteoarthritis. Ming Chen, et al  found that the synovial fluid TGF-β1 level in patients with knee osteoarthritis (KOA) belonged to a significantly negative correlation with the KOA stages. The synovial fluid TGF-β1 level in patients with KOA at middle and advanced stages was significantly lower than that in the control group (P < 0.05), compared to the healthy control group, the synovial fluid TGF-β1 level in patients with KOA at early stage was not statistically significant (P < 0.05). The aforesaid studies showed that TGF-β1 might protect and repair the knee osteoarthritis (KOA) and it was an indicator to measure the severity of the diseases. In this study, PKOA was divided into the mild and severe PKOA. The experiments concluded that the differences were not statistically significant after the comparison of genotype and allele frequency in the two groups, and -509C/T gene polymorphism was not associated with the severity of PKOA.
The difference of the aforesaid research findings might be related to the quantity & size of samples, different regions, ethic groups, and genetic heterogeneity of the studied populations, because the generation of PKOA is a complicated pathological process of multi-gene inheritance. The genetic heterogeneity, environment factors, interaction between environment factors, genetic factors, and other complicated factors might affect the PKOA. Therefore, the interactions among genes and between genes and environment have yet to be further studied.
This work was supported by grants from the Science & Technology Program of the Department of Science and Technology of Hunan Province (program number: 2013FJ3152) and the program of Hunan Academy of Traditional Chinese Medicine (program number: 201224).
The authors declare no conflict of interests.