ESRRBpolymorphisms are associated with comorbidity of temporomandibular disorders and rotator cuff disease

Abstract

Temporomandibular disorders (TMD) are associated with comorbidity. Shoulder pain is among the symptoms associated with TMD. The purpose of this study was to investigate the association between TMD and rotator cuff disease (RCD) and related genetic aspects. All subjects underwent orofacial and shoulder examinations. The control group comprised 30 subjects with no pain. Affected subjects were divided into three groups: RCD (TMD-free, n = 16), TMD (RCD-free, n = 13), and TMD/RCD (patients with both RCD and TMD, n = 49). A total of eight single nucleotide polymorphisms in the ESRRB gene were investigated. A chemiluminescent immunoassay was used to measure estradiol levels. Surface electromyography recorded head and cervical muscle activity. The χ 2 test and Student t -test/Mann–Whitney test were used to assess the significance of nominal and continuous variables. A P -value of <0.05 was considered significant. TMD subjects were seven times more susceptible to RCD than controls. The rs1676303 TT ( P = 0.02) and rs6574293 GG ( P = 0.04) genotypes were associated with RCD and TMD, respectively. TMD/RCD subjects showed associations with rs4903399 ( P = 0.02), rs10132091 ( P = 0.02), and CTTCTTAG/CCTCTCAG ( P = 0.01) haplotypes and lower muscle activity. Estradiol levels were similar among groups. This study supports TMD as a risk factor for RCD. ESRRB haplotypes and low muscle activity are common biomechanical characteristics in subjects with both diseases.

Temporomandibular disorders (TMDs) are the most common source of non-odontogenic pain of musculoskeletal origin. These disorders affect the temporomandibular joint (TMJ) and masticatory muscles. TMDs are heterogeneous in presentation and multifactorial in aetiology. However, it has been hypothesized that persistent TMD pain conditions result from a ‘central sensitization syndrome’, disregarding other important etiological factors, such as trauma, pro-inflammatory states, and a genetic basis.

The overlap of physical symptoms of TMD with those of other comorbid disorders involving pain in the muscles and joints has recently been reported. The facial pain may radiate to surrounding areas triggering jaw pain, earache, tinnitus, headache, cervical/shoulder pain, neuralgia, and toothache. Among these, shoulder pain is one of the main symptoms in TMD patients. However, the most common cause of chronic shoulder pain in adults is rotator cuff disease (RCD), which is a spectrum of disorders varying from reversible tendinopathy to frank tear, affecting 30–50% of the population. The specific aetiology of RCD has not been fully elucidated, but it is considered to be the result of articular degeneration, hypovascularity, collagen abnormalities, tensile overload, and genetic factors – all common to the development of TMD.

The mechanical aetiology of chronic facial and shoulder pain has been related to poor posture of the head–neck–shoulder complex. However, the masticatory and cervical muscle activities in patients with TMD associated with RCD have not been studied previously in order to elucidate the mechanical basis of these comorbid pain conditions.

Epidemiological data have shown that women are predominantly affected by RCD and TMD. This gender difference could be explained on the basis of sex hormones and their receptors. In humans, 17β-estradiol decreases sensitivity to noxious subcutaneous stimuli over the TMJ region. Low oestrogen or rapid changes in oestrogen concentration result in an increase in articular pain, explaining the greater pain intensity observed in women with TMD and RCD.

Endogenous oestrogen can act directly on monocytes, increasing the production of pro-inflammatory cytokines, which promotes cartilage resorption, inhibits the synthesis of proteoglycans, and causes inflammation. This hormone can also increase type III collagen content and lead to a decrease in the type I/III collagen ratio, affecting the healing process.

Traditionally, it has been thought that oestrogen acts only through oestrogen receptors α and β. However, another subfamily within the nuclear receptor subfamily – the oestrogen-related receptors (ERRs) – shares sequence similarity, co-regulatory proteins, and action sites with oestrogen receptors. This subfamily contains three members: ERR α, β, and γ. The oestrogen-related receptor β (ESRRB) is involved in oestrogen-regulated pathways because it can bind the oestrogen response element, activate transcription independent of exogenous ligands, and share co-activators with oestrogen receptors α and β.

There is evidence that genetic factors act as intrinsic risk factors for RCD. In a recent report, different mutations, single nucleotide polymorphism (SNP) functions, and haplotypes of the ESRRB gene were associated with RCD. However, genetic effects on TMJ derangement have not been fully clarified. Since oestrogen alterations are associated with TMD, an investigation of the ESRRB gene may help to gain insights into the pathogenesis of TMD and explore its correlation with RCD.

Taking into account that TMD and RCD are common multifactorial diseases modulated by numerous biological processes, it was hypothesized that the aetiology of TMD/RCD comorbidity is influenced by mechanical muscle activity, oestrogen levels, and the ESRRB gene. Therefore, the purpose of this study was to investigate the association between TMD and RCD comorbidity symptoms and the biomechanical basis. Once a combined diagnosis of TMD and RCD is made, treatment options must be considered. Greater knowledge of these comorbid diseases may help in the identification of therapeutic targets and procedures, providing better strategies to optimize the outcomes of RCD and TMD therapies.

Materials and methods

Subject selection

This cross-sectional study was conducted in accordance with the recommendations of the Ethics Committee of the National Institute of Traumatology and Orthopedic Research; informed consent was obtained from each subject. One hundred eight Brazilian volunteers, of both sexes, were selected from an outpatient pool during the course of 1 year. Subjects reported their personal and medical histories. They underwent routine consultations in a specialized care center for shoulder and elbow disorders in order to evaluate their shoulder and TMJ conditions. Inclusion criteria for subjects were the following: Brazilian citizen, age >45 years, and no previous surgery or neoplasm in the TMJ or shoulder.

Subjects with a history of trauma, bursitis, rheumatoid arthritis, or autoimmune diseases, chronic use of systemic corticosteroids, hyperlaxity, or who were pregnant were excluded. The control group comprised 30 subjects without pain and with no signs or symptoms of TMD or RCD. Subjects diagnosed as having RCD and/or a TMD were divided into three groups: RCD subjects (TMD-free, n = 16), TMD subjects (RCD-free, n = 13), and TMD/RCD affected subjects (patients with both RCD and TMD, n = 49). The baseline clinical parameters for the subject population are shown in Table 1 .

Table 1
Baseline clinical parameters of the population studied.
Variables Control ( n = 30) RCD ( n = 16) TMD ( n = 13) TMD/RCD affected ( n = 49) Healthy TMJ with RCD P -value (OR; CI) a TMD without RCD P -value (OR; CI) a TMD/RCD affected P -value (OR; CI) a
Ethnic group, n (%)
White 11 (36.7) 13 (81.2) 11 (84.6) 38 (77.5) 0.03 (7.4; 1.4–42.7) 0.003 (9.5; 1.5–46.3) 0.0002 (5.9; 1.9–18.5)
Non-white 19 (63.3) 3 (18.7) 2 (15.4) 11 (22.4)
Age, years, mean ± SD 55 ± 7.8 57.2 ± 8.2 56.3 ± 8.1 57.3 ± 8.0 0.06 0.4 0.57
Sex, n (%)
Female 22 (73.3) 11 (68.7) 9 (69.2) 38 (77.5) 0.74 (0.8; 0.1–3.6) 0.7 (0.8; 0.1–4.3) 0.67 (1.26; 0.3–4.0)
Male 8 (26.7) 5 (31.2) 4 (30.8) 11 (22.4)
Smoking, n (%)
Non-smoking 26 (86.7) 13 (81.2) 13 (100) 44 (89.8) 0.6 (0.6; 0.1–4.5) 0.1 (–) 0.67 (1.3; 0.2–6.5)
Smoking 4 (13.3) 3 (18.7) 0 (0) 5 (10.2)
Alcohol consumption, n (%)
Non-drinking 21 (70) 13 (81.2) 11 (84.6) 36 (73.5) 0.4 (1.8; 0.3–10.6) 0.3 (2.3; 0.3–19.0) 0.7 (1.1; 0.3–3.6)
Drinking 9 (30) 3 (18.7) 2 (15.4) 13 (26.5)
General medical condition, n (%)
Systemic disease 30 (100) 16 (100) 13 (100) 46 (93.9) 0.3 1.0 0.1 (0.0; 0.0–3.7)
Diabetes 5 (16.7) 2 (12.5) 5 (38.5) 10 (20.4) 0.7 (0.7; 0.0–5.1) 0.1 (3.1; 0.5–17.5) 0.6 (1.2; 0.3–4.9)
High blood pressure 20 (66.7) 5 (31.2) 5 (38.5) 32 (65.3) 0.02 (0.2; 0.0–0.9) 0.08 (0.3; 0.0–1.4) 0.9 (0.9; 0.3–2.7)
Hypothyroidism 1 (3.3) 2 (12.5) 1 (7.7) 1 (2.0) 0.2 (4.1; 0.2–127.0) 0.5 (2.4; 0.0–98.2) 0.7 (0.6; 0.0–23.1)

RCD, rotator cuff disease; TMD, temporomandibular disorder; OR, odds ratio; CI, confidence interval; SD, standard deviation.

a P -value compared to control group (without either disease).

Diagnosis of temporomandibular disorders

All participants were examined clinically by the same dentist (L.L.B.) according to the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) Axis I in order to investigate the three groups of TMD: group I, muscle disorder; group II, disc displacement; and group III, arthralgia, arthritis, and arthrosis. The RDC/TMD were used to assess these three groups of TMD using well-validated techniques, including palpation at 20 specified muscle sites. Self-reported symptoms pertaining to jaw impairment and associated pain were also recorded during the evaluation.

Clinical characteristics were noted, which included the following clinical symptoms: ear pain, toothache, burning sensation in the mouth, limited mouth opening, and noises (clicking, crepitation) in the TMJ, as well as diagnosed bruxism. Of the 108 patients examined, 62 showed a TMD.

Diagnosis of rotator cuff disease

Sixty-five subjects were diagnosed with RCD. The diagnosis of RCD was based on the protocol of Motta et al., by clinical examination and imaging of the involved shoulder (radiography and magnetic resonance imaging). Subjects considered to be RCD-free had a negative history for shoulder pain, a negative specific test result for impingement syndrome in a complete physical examination of the shoulders, and an absence of tendinopathy. All clinical evaluations were performed by one of the authors from the specialized care center (M.V.A.).

The records of the orthopedist who carried out the RCD diagnosis and the dentist performing the orofacial examination were independent of one another.

Oestrogen-related receptor β genotyping

DNA from all participants was extracted from buccal cells after vigorously rinsing with 5 ml of saline solution for 60 s, as described previously. A NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA) was used to determine the amount and purity of the DNA. Only DNA samples showing A 260 nm /A 280 nm ratios greater than 1.9 were used.

All procedures included in SNP selection and analysis followed the STREGA reporting recommendations. Linkage disequilibrium relationships and gene structure were considered in order to select the eight SNPs in the ESRRB gene included in this study. The minor allele frequencies reported in the database of the National Center for Biotechnology Information ( ) was >0.12 ( Table 2 ).

Table 2
Genetic variants in the ESRRB gene.
SNP Chromosome Base pair position Base change a MAF b SNP type Nearest gene locus
rs4903399 Ch14 76775202 C>T 0.1 Intergenic Intergenic
rs1077430 Ch14 76897677 C>T 0.3 Intron ESRRB
rs2860216 Ch14 77006008 C>T 0.3 Intergenic Intergenic
rs10132091 Ch14 76870818 C>T 0.4 Intron ESRRB
rs1676303 Ch14 76992164 C>T 0.2 Intergenic Intergenic
rs745011 Ch14 76917275 C>T 0.4 Intron ESRRB
rs4903419 Ch14 76984655 A>G 0.1 Intergenic Intergenic
rs6574293 Ch14 76870600 A>G 0.1 Intron ESRRB

SNP, single nucleotide polymorphism.

a Base change according to Applied Biosystems.

b MAF: minor allele frequency according to GenBank.

Real-time polymerase chain reactions with TaqMan chemistry (Applied Biosystems, Foster City, CA, USA) were used for genotyping of the eight selected SNPs in a thermocycler (PTC-225 Tetrad, Peltier Thermal Cycler; Bio-Rad Life Science, Corston, UK), yielding 1.5 ml/reaction in total. The haplotype analyses were also considered in order to explore SNP associations.

Estradiol level

Endogenous estrogens are a group of steroidal compounds including 17β-estradiol, estrone, and estriol. Because the bioactivity of 17β-estradiol is the greatest of the three in vivo, it was chosen to evaluate the estradiol concentration in serum.

One 5-ml blood sample was taken from 41 subjects in the total sample group after 8 h of overnight fasting (eight controls, nine RCD, four TMD, and 20 TMD/RCD affected subjects). The blood was centrifuged and the serum samples stored at −20 °C. In order to exclude the influence of hormone variables on estradiol levels in the women, the use of contraceptives, hormone replacement therapy, menopause status, and menstrual cycle status (normal length menstrual cycles (26–35 days duration) or luteal phase of the menstrual cycle (22–25 days based purely on self-report of the first day of menses)) were recorded. For pre-menopausal women, blood was taken only within the first 7 days from the first day of menstrual flow, since during this period the oestrogen level is not under the influence of the physiology of menstruation.

Assays were performed concurrently on serum specimens from cases and controls. All laboratory personnel were blinded to the subjects’ status. A competitive chemiluminescent immunoassay performed using the ACS-180 automated immunoassay system (Bayer Diagnostics Corp., Tarrytown, NY, USA) was used to measure total estradiol in the serum samples.

The normal values for estradiol were determined according to the manufacturer’s instructions: menopause, <40 pg/ml; non-pregnancy, 27–433 pg/ml; and men, <47 pg/ml. Taking into account the hormonal influence and the natural difference between men and women for serum estradiol levels, subjects were classified as having a normal level, below normal level, or above normal level.

Surface electromyography

The activity of the muscles was recorded by surface electromyography (EMG) on the right and left side masseter, anterior temporal, sternocleidomastoid, trapezius, and deltoid muscles in a representative sample, including 12 subjects from the control and TMD/RCD affected groups. EMG signals were obtained using a four-channel module (EMG System do Brasil Ltda, São José dos Campos, SP, Brazil). Surface active bipolar electrodes with 10 mm of inter-electrode distance and Ag 99.9% (3 M Brazil, Sumaré, São Paulo, Brazil) were used, along with a disposable monopolar electrode, used as a reference (Ag/AgCl; 3 M Brazil) and placed on the forearm.

Patients were instructed to remain seated in a chair, feet apart, shoulders relaxed, and hands resting on their thighs, with their heads in the Frankfort plane parallel to the ground. The electrode attachment sites were cleaned with a cotton ball soaked in 70% alcohol to reduce the impedance between the skin and electrodes, as per the SENIAM (Surface ElectroMyoGraphy for the Non-Invasive Assessment of Muscles project) recommendations. EMG signals were sampled at 1000 Hz, band-pass from 20 to 500 Hz. The signals were recorded for 10 s each. In all tests, a 5-s period was selected (the two initial and three final seconds of the EMG signal were discarded). The signal processing was performed using specific routines carried out in WinDaq software (WinDaq/HS; DATAQ Instruments Inc., Akron, Ohio, USA) and was based on the protocol of Lauriti et al., calculating the average value and standard deviation from the root mean square (RMS).

Masseter and anterior temporal muscles

Evaluations of the masseter and anterior temporal muscles were carried out at rest and at maximum clenching effort (MCE). To avoid direct occlusal contact, a strip of Parafilm M (Bemis Company, Inc., Oshkosh, WI, USA) was folded five times and arranged bilaterally in the molar region, based on the protocol of Lauriti et al.

Sternocleidomastoid muscle

EMG of the sternocleidomastoid muscle was recorded by placing an electrode along a line drawn from the sternal notch to the mastoid process, at one-third the distance from the mastoid process. Subjects were asked to turn their head and neck to maximum rotation.

Trapezius muscle

EMG of the upper trapezius muscle was recorded by placing an electrode along a line joining the acromion and C7, at one-third the distance from the acromion process. These steps were based on a protocol by Chowdhury et al. Subjects had to raise their shoulders to their maximum position in order to analyze trapezius activity.

Deltoid muscle

EMG activity in the deltoid muscle was measured under complete arm abduction with electrodes positioned on the greatest tonus.

Statistical analysis

Data entry and the statistical analysis were implemented with Stata 11.1 (StataCorp, College Station, TX, USA). The sample included all subjects attending for routine consultation at the Center of Shoulder and Elbow Surgery of the National Institute of Traumatology and Orthopedics who fulfilled the inclusion criteria. The χ 2 test was used to assess the significance of differences in nominal variables (expressed as frequencies and percentages), including the frequencies of genotypes and alleles, between cases and controls. After the Shapiro–Wilk test for continuous variables was applied (variables expressed as the mean and standard deviation), analysis of variance was performed with the Student t -test or Mann–Whitney test, depending on whether the distribution was normal or non-normal, respectively. All genetic analyses were performed just after fitting for Hardy–Weinberg equilibrium. The odds ratio (OR) and 95% confidence interval (95% CI) were used to calculate the risk associated with alleles and genotyping. Multivariate logistic regression explored variables with statistical significance in the univariate analysis. Multiple comparisons were corrected by Bonferroni correction ( www.quantitativeskills.com/sisa/calculations/bonfer.htm ). The program ARLEQUIN (v.20; anthro.unige.ch/arlequin ) was used to calculate linkage disequilibrium and haplotypes. Values of P < 0.05 were considered to have statistical significance.

Results

Clinical findings: TMD and RCD incidence

Of the total 410 subjects evaluated during 1 year at the specialized care center for shoulder and elbow disorders, 108 were included in this study. There were 80 (74%) women and 28 (26%) men, with a mean age of 57.2 ± 8.3 years. The control group consisted of 30 subjects, 22 (73%) women and eight (27%) men, with a mean age of 55 ± 7.8 years. No difference was found among test groups (RCD, TMD, and TMD/RCD affected) when compared to the control group for age, sex, smoking habit, alcohol consumption, systemic disease, diabetes, hypothyroidism, and the use of calcium supplementation, analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), bisphosphonates, and muscle relaxant drugs.

The χ 2 test revealed a higher prevalence of white ethnicity in the TMD ( P = 0.003), RCD ( P = 0.03), and TMD/RCD affected ( P < 0.001) groups. On the basis of OR calculation, the risk associated with those of white ethnicity having both diseases (OR 5.9; 95% CI 1.9–18.5) was six times higher than in control subjects. Additional details regarding the demographics of the subjects are shown in Table 1 .

During the clinical TMJ and shoulder evaluations on the subjects, the incidence of TMD was 57.4% and RCD was 60.2%. From the total patients with TMD ( n = 62), 79.0% ( n = 49) were diagnosed with RCD. The χ 2 Fisher’s test showed a marked difference in RCD incidence in patients with and without TMD ( P < 0.001). Subjects with TMD were seven times more susceptible to RCD (OR 7.0; 95% CI 2.7–18.4) than subjects with a healthy TMJ.

No difference was detected among the groups with TMD according to the RDC/TMD Axis I diagnosis. From the total of 62 subjects diagnosed with TMD, 24 showed a muscle disorder (group I), three had disc displacement (group II), and five were classified with a degenerative local disease (group III); more than one diagnosis was identified in 30 of the subjects ( Table 3 ).

Table 3
Characteristics of all subjects with TMD according to the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) Axis I evaluation.
With TMD ( n = 62) P -value OR 95% CI
Without RCD
( n = 13)
With RCD
( n = 49)
Muscle disorders (group I) 7 17 0.2 0.4 0.1–1.8
Disc displacement (group II) 1 2 0.5 0.5 0.0–15.5
Arthralgia, arthritis, and arthrosis (group III) 0 5 0.2
Multiple diagnoses 5 25 0.4 1.6 0.4–6.9
TMD, temporomandibular disorder; RCD, rotator cuff disease; OR, odds ratio; CI, confidence interval.

Genetic association study

The results for the eight SNPs in the ESRRB gene are provided in Table 4 . As indicated by the χ 2 test, statistically significant associations were observed. Allele and genotype frequencies for SNPs were within Hardy–Weinberg equilibrium in all groups. All evaluations considered the control group as the reference for statistical calculation.

Table 4
ESRRB genotype and allele distribution in all subjects.
SNP Genotypes Control ( n = 30) RCD ( n = 16) TMD ( n = 13) TMD/RCD affected ( n = 49) Healthy TMJ with RCD P -value (OR; CI) a TMD without RCD P -value (OR; CI) a TMD/RCD affected P -value (OR; CI) a
rs4903399 CC–CT–TT 14–13–2 14–2–0 6–6–1 36–12–1 0.031 0.9 0.07
CT+TT 15 2 7 13 0.009 (0.1; 0.0–0.8) 0.8 (1.0; 0.24–4.91) 0.02 (0.3; 0.1–0.9)
C 41 30 18 84 0.01 (6.2; 1.2–42.2) 0.2 (2.3; 0.5–11.6) 0.02 (2.4; 1.0–5.9)
T 17 2 3 14
rs1077430 CC–CT–TT 6–14–9 4–9–3 2–5–6 11–21–17 0.6 0.6 0.8
CT+TT 23 12 11 38 0.5 (0.7; 0.1–4.1) 0.6 (1.4; 0.2–12.3) 0.8 (0.9; 0.2–3.1)
C 26 17 9 43 0.4 (1.3; 0.5–3.6) 0.3 (0.6; 0.2–1.8) 0.9 (0.9; 0.4–1.9)
T 32 15 17 55
rs2860216 CC–CT–TT 3–14–12 3–4–9 2–8–3 4–16–29 0.2 0.5 0.3
CT+TT 26 13 11 45 0.4 (0.5; 0.0–3.7) 0.6 (0.6; 0.0–6.4) 0.7 (1.3; 0.2–7.6)
C 20 10 12 24 0.7 (0.8; 0.3–2.3) 0.3 (1.6; 0.5–4.6) 0.1 (0.6; 0.2–1.3)
T 38 22 14 74
rs10132091 CC–CT–TT 8–16–5 4–7–5 2–5–6 4–25–20 0.5 0.1 0.02
CT+TT 21 12 11 45 0.8 (1.1; 0.2–5.7) 0.3 (2.1; 0.3–17.2) 0.02 (4.2; 1.0–19.4)
C 32 15 9 33 0.4 (0.7; 0.2–1.8) 0.08 (0.4; 0.1–1.2) 0.008 (0.4; 0.2–0.8)
T 26 17 17 65
rs1676303 CC–CT–TT 1–7–21 2–9–5 1–1–11 4–17–28 0.02 0.4 0.3
CT+TT 28 14 12 45 0.2 (0.2; 0.0–4.0) 0.5 (0.4; 0.0–17.3) 0.4 (0.4; 0.0–4.1)
C 9 13 3 25 0.007 (3.7; 1.2–11.4) 0.6 (0.7; 0.1–3.2) 0.1 (1.8; 0.7–4.7)
T 49 19 23 73
rs745011 CC–CT–TT 9–13–7 7–9–0 3–2–8 12–26–11 0.09 0.05 0.7
CT+TT 20 9 10 37 0.3 (0.5; 0.1–2.4) 0.5 (1.5; 0.2–8.9) 0.5 (1.3; 0.4–4.3)
C 31 23 8 50 0.0 (2.2; 0.8–6.2) 0.5 (0.3; 0.1–1.1) 0.7 (0.9; 0.4–1.8)
T 27 9 18 48
rs4903419 AA–AG–GG 20–8–1 13–3–0 9–4–0 32–17–0 0.5 0.7 0.3
AG+GG 9 3 4 17 0.3 (0.5; 0.0–2.6) 0.9 (0.9; 0.1–4.9) 0.7 (1.1; 0.4–3.5)
A 48 29 22 81 0.3 (2.0; 0.4–10.1) 0.8 (1.1; 0.2–4.9) 0.9 (0.9; 0.3–2.5)
G 10 3 4 17
rs6574293 AA–AG–GG 0–11–18 0–5–11 0–1–12 0–10–39 0.6 0.04 0.09
AG+GG 29 16 13 49
A 11 5 1 10 0.6 (0.7; 0.2–2.8) 0.06 (0.1; 0.0–1.4) 0.1 (0.4; 0.1–1.3)
G 47 27 25 88
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Jan 16, 2018 | Posted by in Oral and Maxillofacial Surgery | Comments Off on ESRRBpolymorphisms are associated with comorbidity of temporomandibular disorders and rotator cuff disease
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