Abstract
The aim of this study was to evaluate the association between genetic polymorphisms and the comorbid presence of chronic systemic arthralgia in patients with articular temporomandibular disorders (TMD). Subjects were evaluated for the presence of TMD and asked about the presence of chronic joint pain. Four groups were included in the study: articular TMD and systemic arthralgia ( n = 85), no articular TMD and systemic arthralgia ( n = 82), articular TMD and no systemic arthralgia ( n = 21), no articular TMD and no systemic arthralgia (control, n = 72). A total of 14 single nucleotide polymorphisms in the OPG , RANK , and RANKL genes were investigated. In the statistical analysis, a P -value of <0.05 was considered significant. For the OPG gene, an association was observed between the group with chronic arthralgia and joint TMD and the control group ( P = 0.04). There was also a tendency towards an association of the haplotype CGCCAA with an increased risk of developing chronic joint pain, even in the absence of TMD ( P = 0.06). For the RANK gene, the AGTGC haplotype was associated with the lowest risk of presenting chronic joint pain in individuals without TMD ( P = 0.03). This study supports the hypothesis that changes in the OPG and RANK genes influence the presence of chronic joint pain in individuals with and without TMD.
Osteoarthritis (OA) of the temporomandibular joint (TMJ) is a degenerative disease characterized by low intensity inflammation , which generally begins with synovitis and progressive cartilage degradation, with subsequent remodelling of the subchondral bone. This usually leads to limited mandibular function and chronic pain . Although the degradation of articular cartilage is one of the main characteristics of OA, the precise mechanism remains unknown .
Recent studies have reported that damage to the subchondral bone in hyaline cartilage joints, resulting from changes in the induction, proliferation, and metabolism of osteoblasts and osteoclasts, may be responsible for the onset and progression of cartilage degeneration . During the OA process, the subchondral bone undergoes metabolic and structural alterations, which lead to microfractures, angiogenesis, and subsequently to reactive bone sclerosis. Histologically, the tidemark (line that divides the calcified and uncalcified cartilage) is duplicated, and there is penetration of new blood vessels into the calcified cartilage and an increase in subchondral bone thickness . Such changes affect the biomechanical properties of overlying articular cartilage and its biological relationship with the subchondral bone .
These distinct responses to the OA process are influenced by the expression of different genes that are responsible for the control of communication between osteoblasts and osteoclasts, fundamental to the balance and maintenance of joint structure and function . The key signalling pathway that directly influences this communication involves molecular receptors such as receptor activator of nuclear factor kappa B (RANK), its ligand (RANKL), and osteoprotegerin (OPG), more formally known as tumour necrosis factor receptor superfamily member 11 B (TNFRSF11B) .
In a study by Wakita et al., a marked decrease in the concentration of OPG was detected in the synovial fluid of patients with internal derangements of the TMJ . Treatment of the synovial fluid in patients with temporomandibular OA resulted in an elevated production of cells with osteoclastic characteristics from the mononuclear cells in vitro. The addition of anti-RANKL immunoglobulin or OPG attenuated osteoclast formation induced by this means, suggesting that an increase in the RANKL to OPG ratio in the articular microenvironment has the potential to induce osteoclastogenesis in the TMJ. In another study, the induction of psychological stress and abnormal mechanical articular overload in animal specimens resulted in subchondral bone loss in the mandibular condyles . The pathophysiology of this process was found to involve an increase in the secretion of RANKL by adult mesenchymal cells from condylar bone marrow, thus exacerbating osteoclastic activity.
It has already been shown that polymorphisms in regions of the regulatory genes of the RANK/RANKL/OPG receptors are associated with joint destruction in patients with rheumatoid arthritis stress fractures of the bones in elite athletes (rs3018362 (RANK) and rs1021188 (RANKL)) , and alterations in remodelling, bone fractures, and bone mineral density in pre- and post-menopausal women .
It is estimated that approximately 50% of temporomandibular OA cases are related to genetic polymorphisms involving the genes related to the metabolism of chondrocytes, osteoblasts, and osteoclasts . However, to date, no controlled studies have evaluated genetic alterations in the osteoclastogenesis system in individuals with TMJ disorders (TMD).
It is believed that the development of OA, both in the TMJ and in other joints in the body, does not occur randomly. The hypothesis raised in this study was that there is a genetic basis for the comorbid clinical development of both conditions. The presence of polymorphisms in genes associated with osteoblast and chondroblast metabolism could be related to the development of joint disease. Thus, the aim of this study was to determine the possible association between polymorphisms in the RANK , RANKL , and OPG genes, and the combined presence of chronic systemic arthralgia in individuals with TMD.
Materials and methods
This was a descriptive, cross-sectional, randomized study, approved on May 30, 2013 by the Research Ethics Committee of University Salty Olive − Universe. Free and informed consent was obtained from participants in writing prior to conducting the research. The recommendations of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement were followed during the design and development of the study .
The study included individuals attending Fluminense Federal University − either patients seeking care or their companions. All participants had to be between 18 and 65 years of age, with no history of macrotrauma to the TMJ and/or the knee, hip, ankle, shoulder, wrist, and elbow joints. The individuals were recruited at random over a period of 2 years (2013–2015). Exclusion criteria were joint surgery, a diagnosis of rheumatoid arthritis, systemic lupus erythematosus, fibromyalgia, or other types of systemic joint disease, and/or previous treatment for TMD.
The study methodology was divided into three stages, as outlined below.
Stage 1: clinical diagnosis of TMD
All participants were examined clinically by the same evaluator using the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) , as described previously . This process occurs in a manner that is not mutually exclusive, allowing each participant to belong to more than one diagnosis subgroup at the same time.
The joint diagnoses (subgroups II and III) were combined to form a single group called ‘joint disorders’. Thus, two groups were formed: (1) control (no TMD diagnosed); (2) patients with an articular type TMD. A single trained examiner (L.L.B.) used this tool for all patients.
Stage 2: assessment of the presence and duration of other arthralgia
In this stage, each participant completed a questionnaire considering the following: (1) the presence of pain in the knee, hip, ankle, shoulder, wrist, and/or elbow joints, and (2) the duration of the pain (days, months, or years). The questionnaire was administered exclusively by a second examiner (V.Q.), who had no prior knowledge of the presence or absence of TMD in the participants being assessed. The presence of arthralgia for more than 3 months was considered chronic .
According to the diagnosis of TMD and the diagnosis of other arthralgia, the study volunteers were again divided, this time into four groups: (1) those with articular TMD and chronic pain in other joints; (2) individuals without TMD and with chronic systemic arthralgia; (3) individuals with articular TMD and without chronic systemic arthralgia, and (4) individuals without TMD or any type of joint pain (control group).
Stage 3: genotyping
Saliva samples were obtained from all participants and genomic DNA was extracted as described previously . The concentration and purity of the DNA were analyzed using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). All samples had to present an A260 nm/A280 nm ratio greater than 1.9 .
Six single nucleotide polymorphisms (SNPs) in the OPG gene (rs11573919, rs11573875, rs11573854, rs11573838, rs11573817, and rs11573816), five in the RANK gene (rs474369, rs9498322, rs504762, rs6920383, and rs237033), and three in the RANKL gene (rs492956, rs13215304, and rs12660731) were selected, considering the linkage disequilibrium relationships and gene structure. These SNPs were identified previously and included in the National Center for Biotechnology Information (NCBI) database ( ), with the lowest allelic frequency having to be >0.12. All procedures followed the STREGA recommendations (Strengthening the REporting of Genetic Association studies) .
Data processing and the statistical analysis were performed using Stata 12.0 (Stata Corp., College Station, TX, USA). The sample size was calculated on the basis of the spontaneous recruitment of patients and companions at Fluminense Federal University over a period of 2 years, respecting the inclusion criteria. This calculation was designed to detect a risk factor that reached 40% of the sample, with alpha of 5% and 90% power. To verify a normal distribution of the numerical variables, the Shapiro–Wilk test was used, followed by analysis of variance (ANOVA) with the Student t -test and Mann–Whitney test for normal and non-normal distributions, respectively. The χ 2 test was used to evaluate the significance of nominal variables between groups.
Differences in the frequencies of genotypes and alleles between the groups were analyzed using the χ 2 test, after fitting for Hardy–Weinberg equilibrium. Values of P < 0.05 were considered statistically significant, and the risks associated with individual alleles and genotypes were calculated as the odds ratio (OR) with the 95% confidence interval (CI). To calculate linkage disequilibrium and haplotypes, the ARLEQUIN computer program was used (v.3.1; http://cmpg.unibe.ch/software/arlequin3/ ).
Results
Clinical findings: prevalence of chronic arthralgia by age group
From a total of 337 volunteers evaluated during the 2-year period, 260 were included in the study. Of these, 85 had chronic arthralgia and concomitant articular TMD (52 women (61.2%) and 33 men (38.8%)), 82 had chronic arthralgia but were without any TMD diagnosis (52 women (63.4%) and 30 men (36.6%)), and 21 had an articular TMD but were without chronic arthralgia (14 women (66.7%) and seven men (33.3%)). The control group comprised 72 individuals who were free of any joint symptoms and other TMD diagnoses (39 women (54.2%) and 33 men (45.8%)).
Among the locations of reported arthralgia, the knee was the most prevalent in both groups, followed by the hip and shoulder regions ( Table 1 ). No statistically significant difference was found between the two groups studied (articular TMD vs. no TMD) in the prevalence of the sites affected by reported joint pain (χ 2 test).
| Site | With articular TMD ( n = 85), n (%) | Without TMD ( n = 82), n (%) | P -value (OR, 95% CI) |
|---|---|---|---|
| Knee | 59 (69.4) | 48 (58.5) | P = 0.19 (1.6, 0.8–3.0) |
| Hip | 12 (14.1) 73 | 20 (24.4) 62 | P = 0.13 (0.5, 0.2–1.1) |
| Ankle | 4 (4.7) 81 | 5 (6.1) 77 | P = 0.47 (0.7, 0.19–2.9) |
| Shoulder | 7 (8.2) 78 | 9 (11.0) 73 | P = 0.74 (0.7, 0.2–2.0) |
| Wrist | 1 (1.2) 84 | 3 (3.7) 79 | P = 0.29 (0.3, 0.03–3.0) |
| Elbow | 2 (2.4) 82 | 3 (3.7) 79 | P = 0.48 (0.6, 0.1–3.9) |
Genetic association analysis
The characteristics of the 14 polymorphisms studied in the OPG (rs11573919, rs11573875, rs11573854, rs11573838, rs11573817, and rs11573816), RANK (rs474369, rs9498322, rs504762, rs6920383, and rs237033), and RANKL (rs492956, rs13215304, and rs12660731) genes are presented in Table 2 . The frequencies of the genotypes and alleles were adjusted for age, and are provided in Table 3 .
| Gene symbol | Gene name | SNP | Chromosome | Base pair position a | SNP type | Base change b | MAF c | |
|---|---|---|---|---|---|---|---|---|
| Major | Minor | |||||||
| OPG | Tumour necrosis factor receptor superfamily member 11B | rs11573919 | 8 | 118929541 | Intragenic | A | G | 0.02 |
| rs11573875 | 118939606 | Intragenic | C | T | 0.02 | |||
| rs11573854 | 118942974 | Intragenic | A | G | 0.007 | |||
| rs11573838 | 118945400 | Intragenic | A | G | 0.05 | |||
| rs11573817 | 118949504 | Intragenic | C | T | 0.001 | |||
| rs11573816 | 118949803 | Intragenic | C | T | 0.01 | |||
| RANK | Tumour necrosis factor receptor superfamily member 11A | rs474369 | 6 | 149322826 | Intragenic | A | T | 0.21 |
| rs9498322 | 149335950 | Intragenic | A | G | 0.19 | |||
| rs504762 | 149351529 | Intragenic | C | T | 0.21 | |||
| rs6920383 | 149356361 | Intragenic | A | G | 0.19 | |||
| rs237033 | 149393013 | Intragenic | A | G | 0.10 | |||
| RANKL | Tumour necrosis factor superfamily member 11 | rs492956 | 6 | 149347391 | Intragenic | A | G | 0.21 |
| rs13215304 | 149378197 | Synonymous codon | A | G | 0.01 | |||
| rs12660731 | 149381090 | Intragenic | C | T | 0.04 | |||
a Base change according to Applied Biosystems.
| Gene | SNP | Genotypes | Control ( n = 72) | Articular TMD and with arthralgia ( n = 85) | No TMD and with arthralgia ( n = 82) | With articular TMD and without arthralgia ( n = 21) | P -value (OR, 95% CI) (adjusted for age) | ||
|---|---|---|---|---|---|---|---|---|---|
| Control vs. articular TMD and with arthralgia | Control vs. no TMD and with arthralgia | Control vs. with articular TMD and without arthralgia | |||||||
| OPG | rs11573919 | CC–CT–TT | 63–4–0 | 79–1–0 | 77–1–0 | 21–0–0 | 0.19 | 0.11 | 0.13 |
| CT + TT | 4 | 1 | 1 | 0 | 0.13 (0.19, 0.02–1.82) | 0.13 (0.2, 0.02–1.8) | 0.32 (−) | ||
| C | 130 | 159 | 155 | 42 | 0.13 (4.8, 0.5–44.3) | 0.14 (4.7, 0.5–43.2) | 0.33 (−) | ||
| T | 4 | 1 | 1 | 0 | |||||
| rs11573875 | AA–AG–GG | 0–1–68 | 0–4–79 | 0–4–75 | 0–0–21 | 0.04 | 0.21 | 0.46 | |
| AG + GG | 69 | 83 | 79 | 21 | (−) | (−) | (−) | ||
| A | 1 | 4 | 4 | 0 | 0.24 (3.3, 0.37–30.6) | 0.23 (3.5, 0.3–32.2) | 0.76 (−) | ||
| G | 137 | 162 | 154 | 42 | |||||
| rs11573854 | CC–CT–TT | 64–4–0 | 77–5–0 | 80–1–0 | 21–0–0 | 0.58 | 0.11 | 0.14 | |
| CT + TT | 4 | 5 | 1 | 0 | 0.61 (1.0, 0.2–4.0) | 0.13 (0.2, 0.2–1.8) | 0.33 (−) | ||
| C | 132 | 159 | 161 | 42 | 0.61 (0.9, 0.25–3.6) | 0.13 (4.8, 0.5–44.1) | 0.33 (−) | ||
| T | 4 | 5 | 1 | 0 | |||||
| rs11573838 | CC–CT–TT | 71–1–0 | 84–1–0 | 82–0–0 | 21–0–0 | 0.95 | 0.22 | 0.47 | |
| CT + TT | 1 | 1 | 0 | 0 | 0.70 (0.8, 0.05–13.7) | 0.46 (−) | 0.77 (−) | ||
| C | 143 | 169 | 164 | 42 | 0.70 (1.1, 0.07–19.0) | 0.46 (−) | 0.77 (−) | ||
| T | 1 | 1 | 0 | 0 | |||||
| rs11573817 | AA–AG–GG | 69–1–0 | 84–1–0 | 79–0–0 | 21–0–0 | 0.97 | 0.22 | 0.47 | |
| AG + GG | 1 | 1 | 0 | 0 | 0.70 (0.82, 0.05–13.3) | 0.46 (−) | 0.76 (−) | ||
| A | 139 | 169 | 158 | 42 | 0.70 (1.21, 0.07–19.6) | 0.46 (−) | 0.76 (−) | ||
| G | 1 | 1 | 0 | 0 | |||||
| rs11573816 | AA–AG–GG | 0–15–49 | 0–12–61 | 0–21–53 | 0–5–10 | 0.32 | 0.51 | 0.44 | |
| AG + GG | 64 | 73 | 74 | 15 | (−) | (−) | (−) | ||
| A | 15 | 12 | 21 | 5 | 0.22 (0.6, 0.3–1.5) | 0.67 (1.2, 0.6–2.5) | 0.32 (1.5, 0.5–4.5) | ||
| G | 113 | 134 | 127 | 25 | |||||
| RANK | rs474369 | AA–AT–TT | 59–5–3 | 79–4–1 | 72–4–5 | 16–5–0 | 0.15 | 0.75 | 0.07 |
| AT + TT | 8 | 5 | 9 | 5 | 0.31 (0.4, 0.14–1.4) | 0.92 (0.92, 0.33–2.5) | 0.16 (2.3, 0.6–8.0) | ||
| A | 123 | 162 | 148 | 37 | 0.13 (2.4, 0.8–6.7) | 0.92 (0.9, 0.4–2.1) | 0.32 (0.6, 0.2–2.0) | ||
| T | 11 | 6 | 14 | 5 | |||||
| rs9498322 | AA–AG–GG | 36–29–6 | 44–37–4 | 40–30–4 | 9–7–3 | 0.8 | 0.75 | 0.67 | |
| AG + GG | 35 | 41 | 34 | 10 | 0.51 (0.9, 0.5–1.8) | 0.8 (0.8, 0.4–1.6) | 1.0 (1.1, 0.4–3.1) | ||
| A | 101 | 125 | 110 | 25 | 0.72 (1.1, 0.8–1.8) | 0.63 (1.1, 0.7–1.9) | 0.66 (0.7, 0.3–1.6) | ||
| G | 41 | 45 | 38 | 13 | |||||
| rs504762 | CC–CT–TT | 0–12–59 | 0–12–70 | 0–13–68 | 0–3–18 | 0.56 | 0.89 | 0.77 | |
| CT + TT | 71 | 82 | 81 | 21 | (−) | (−) | (−) | ||
| C | 12 | 12 | 13 | 3 | 0.88 (0.8, 0.3–1.9) | 0.92 (0.9, 0.42.1) | 0.53 (0.8, 0.2–3.1) | ||
| T | 130 | 152 | 149 | 39 | |||||
| rs6920383 | AA–AG–GG | 38–24–7 | 47–34–2 | 44–27–3 | 8–9–3 | 0.17 | 0.35 | 0.49 | |
| AG + GG | 31 | 36 | 30 | 12 | 0.48 (0.9, 0.4–1.7) | 0.71 (0.8, 0.4–1.6) | 0.35 (1.8, 0.6–5.0) | ||
| A | 100 | 128 | 115 | 25 | 0.42 (1.2, 0.7–2.1) | 0.3 (1.3, 0.7–2.2) | 0.31 (0.6, 0.3–1.3) | ||
| G | 38 | 38 | 33 | 15 | |||||
| rs237033 | CC–CT–TT | 44–24–1 | 51–29–3 | 50–25–2 | 13–8–0 | 0.93 | 0.86 | 0.74 | |
| CT + TT | 25 | 32 | 27 | 8 | 0.88 (1.1, 0.5–2.1) | 0.5 (0.9, 0.4–1.8) | 0.9 (1.0, 0.3–2.9) | ||
| C | 112 | 131 | 125 | 34 | 0.72 (0.8, 0.4–1.5) | 0.88 (1.0, 0.5–1.8) | 0.84 (0.9, 0.4–2.3) | ||
| T | 26 | 35 | 29 | 8 | |||||
| RANKL | rs492956 | AA–AG–GG | 55–12–0 | 66–12–1 | 59–17–0 | 16–3–0 | 0.65 | 0.51 | 0.83 |
| AG + GG | 12 | 13 | 17 | 3 | 0.49 (0.9, 0.3–2.1) | 0.6 (1.3, 0.5–3.0) | 0.56 (0.8, 0.2–3.4) | ||
| A | 122 | 144 | 135 | 35 | 0.86 (1.0, 0.45–2.2) | 0.67 (0.7, 0.3–1.7) | 0.56 (1.1, 0.3–4.2) | ||
| G | 12 | 14 | 17 | 3 | |||||
| rs13215304 | AA–AG–GG | 66–2–0 | 78–3–0 | 72–5–0 | 17–1–0 | 0.93 | 0.31 | 0.61 | |
| AG + GG | 2 | 3 | 5 | 1 | 0.58 (1.2, 0.2–7.8) | 0.27 (2.2, 0.4–12.2) | 0.5 (1.9, 0.1–22.6) | ||
| A | 134 | 159 | 149 | 35 | 0.58 (0.7, 0.13–4.8) | 0.27 (0.44, 0.08–2.3) | 0.5 (0.5, 0.04–5.9) | ||
| G | 2 | 3 | 5 | 1 | |||||
| rs12660731 | CC–CT–TT | 69–1–0 | 84–0–0 | 81–0–0 | 21–0–0 | 0.34 | 0.21 | 0.47 | |
| CT + TT | 1 | 0 | 0 | 0 | 0.45 (−) | 0.46 (−) | 0.76 (−) | ||
| C | 139 | 168 | 162 | 42 | 0.45 (−) | 0.46 (−) | 0.76 (−) | ||
| T | 1 | 0 | 0 | 0 | |||||
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