Osteoprotegerin Gene Polymorphism in Egyptian RA Patients

Introduction

Rheumatoid Arthritis (RA) is an autoimmune disorder that affects 0.5-1% of the population and is associated with significant morbidity, disability, and costs for society(1).

Atherosclerotic disease in both its subclinical and clinically established phases is widely prevalent throughout the world. Disease progression can eventually lead to the occurrence of acute cardiovascular events (CVE), such as myocardial infarction, unstable angina pectoris, and sudden cardiac death(2).

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RA is associated with increased premature mortality mainly due to cardiovascular (CV) diseases (3). Atherosclerosis is emerging as an important complication of RA, with coronary artery disease being the major cause of mortality in these patients. Both men and women with RA are twice as likely to suffer from myocardial infarction when compared with the general population (4).

Subclinical atherosclerosis, mainly carotid artery plaques, may be observed in RA patients, which may be easily recognized by ultrasound, thus identifying those patients with higher CVE risk (5). Multiple lines of evidence reported that CV risk factors are probably underestimated in RA patients (6) , although the international recommendations clearly state about the assessment of this specific risk (7), the evidence of traditional CV risk factors and subclinical atherosclerosis does not fully explain the increased incidence of CVEs in these patients; suggesting that the CV risk may be independently associated with RA and in fact, this risk has been shown to be associated with additional features specific of RA, such as the systemic inflammatory process, disease duration and therapeutic strategies (3, 8-10).

Because heritability accounts for up to 60% of the risk in RA and 30%–60% of the risk in cardiovascular disease,(11) there has been increasing interest in attempting to identify genetic markers of atherosclerosis in RA. Multiple variants in multiple genes have been investigated regarding their association with clinical and subclinical cardiovascular (CV) disease, traditional CV risk factors, and CV mortality(12).

The human Osteoprotegerin (OPG) gene (also called TNFRSF11B) located on chromosome 8q24 is affected by genetic polymorphisms with functional consequences on CV disease and bone metabolism (13, 14). Several groups have reported that a single-nucleotide OPG polymorphism (SNP) located in the 59 UTR region (rs2073617), as well as one in exon 1 (rs2073618) and another in the promoter region (rs3134069), were associated with atherosclerosis and risk of cerebrovascular disease in nonrheumatic individuals (15, 16).

Osteoprotegerin (OPG) is a decoy receptor activator for nuclear factor κB ligand (RANKL) that acts as a regulator of bone resorption, immunity, and cardiovascular function. OPG expression has been detected in human atherosclerotic plaque as well as in osteoblasts (17, 18).

It is secreted by endothelial cells and smooth muscle cells in response to stimulation by proinflammatory cytokines, and it up-regulates the expression of endothelial adhesion molecules that facilitate the migration of monocytes and lymphocytes into the vascular intima during the process of atherogenesis (19).

OPG is also related to plaque rupture (20, 21). OPG is also expressed in synovial tissue obtained from the joints of RA patients suggesting a possible role in the pathogenesis of both atherosclerosis and RA (22).

Subsequently, we evaluated whether polymorphism rs2073618 in the exon I of the OPG gene affects plasma OPG levels and whether this polymorphism is associated with markers of subclinical carotid atherosclerosis in Egyptian Patients with Rheumatoid Arthritis

Patients and methods

Subjects

This cross-sectional study enrolled 80 patients with rheumatoid arthritis who were selected from the outpatient clinics and inpatient department of Rheumatology and Rehabilitation in Assiut University Hospitals.

In our study we had two groups of patients :

1. Group I: RA patients with atherosclerosis (no=38),
2. Group II: RA patients without atherosclerosis (no=42

Inclusion criteria:

• Participants are older than 18 years.
• All patients with RA were diagnosed and fulfilled American College of Rheumatology /European league against] rheumatism (ACR/EULAR) 2010 criteria for RA (23).

Exclusion criteria:

• Patients under the age of 18 years.
• Patients with definite diagnosis for any other systemic autoimmune disorders.
• Patients with a history of cardiovascular disease (previous stroke myocardial infarction or angina)

Clinical assessment

Every patient was subjected to the following:

• A) History taking including Age, sex, disease duration, history of the present illness, drug intake including steroids and calcium, past and family history of cardiovascular events, and risk factors for atherosclerosis (diabetes mellitus, smoking, hypertension, dyslipidemia).
• B) Physical examination including thorough clinical examination including:

1. Height and weight were measured and body mass index ( BMI) of the participants was calculated
2. Blood pressure was determined as the average of 2 measurements obtained 5 minutes apart after subjects had rested in the supine position for at least 10 minutes. Subjects were considered to have hypertension if they were taking antihypertensive agents or according to the latest version of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC-7) recommendations(24).

• C)Activity of RA was defined by Disease Activity Score (DAS28 ) (25)
• D) Ability to perform activities of daily living was measured using the Modified Health Assessment Questionnaire (mHAQ)(26).

Laboratory assessment

including complete blood picture, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), High sensitive CRP(mg/L), plasma glucose, Liver function tests, Kidney function tests, rheumatoid factor titer, and anticyclic citrullinated peptide antibodies. A complete lipid profile was obtained with the measurement of total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides.

Human Osteoprotegerin (OPG) measurement

OPG ELISA (Enzyme-Linked-Immunosorbent Assay), SinoGeneClon Biotech Co Kit was used for the quantitative determination of OPG in serum samples, according to the manufacturer’s instructions.

The kit adopt purified OPG antibody to coat microtiter plate, make solid-phase antibody, was add OPG to wells, Combined OPG antibody with labeled HRP(horse radish peroxidase) to form antibody-antigen –enzyme antibody complex, after washing completely, add TMB(3,3,5,5 tetramethylbenzidin) substrate solution, TMB substrate becomes blue color at HRP enzyme-catalyzed, reaction is terminated by the addition of a stop solution and the color change is measured at a wavelength of 450 nm. The concentration of OPG by ng/L in the samples was determined by comparing the O.D.(optical density) of the samples to the standard curve.

Carotid artery ultrasonography:

The common carotid arteries were carefully examined for wall changes in all subjects, obtaining different longitudinal and transverse views with the high-resolution B-mode ultrasound equipment Logic P6 PRO GE healthcare. A region about 1.5 cm proximal to the carotid bifurcation was identified, and the intima-media thickness (IMT) of the far wall was evaluated as the distance between the luminal-intimal interface and the medial adventitial interface. One transverse and two longitudinal measurements of IMT were obtained from 4 contiguous sites at 2mm intervals, and the average of the 4 measurements were used for the analysis.

According to current sonographic criteria, we referred to ‘‘normal’’ IMT when complex intima-media is (0.9 mm). MIMT values> 0.9 mm were considered indicative of thickened intima (27).

Plaques were defined as focal widening relative to adjacent segments, with protrusion into the lumen of calcified or noncalcified material

All ultrasound measurements were performed by the same examiner who was unaware of the subject characteristics.

All these data were recorded for every patient in a separate sheet

Ethical consideration

The study was approved by the Institutional Ethics Committee Faculty of Medicine Assiut University. Also Informed consent was prepared by Arabic language prepared to be read by the participating patients or to read upon them if they were illiterate and then to be signed.

Statistical analyses

Data was collected and analyzed using SPSS (Statistical Package for the Social Science, version 20, IBM, and Armonk, New York). Continuous data was expressed in form of mean ± SD or median (range) while nominal data was expressed in form of frequency (percentage).

Chi²-test was used to compare the nominal data of different groups in the study while student t-test was used to compare mean of different two groups and ANOVA test for more than two groups in case of normally distributed data while Wilcoxon, Mann-Whitney, and Kruskal Wallis tests were used in case of not- normally distributed data. Person correlation was used to determine the correlation between OPG and other continuous variables.

Results

Demographic Data of Studied Groups:

As observed in Table1 Mean age of patients with rheumatoid arthritis (RA) and atherosclerosis was 56.64 ± 6.43 years, 89.5% of them were females, and 65.7% of them came from rural areas. The mean age of that patient with RA and without atherosclerosis was 51.78 ± 4.97 years, 90.5% of them were female and 64.3% of them came from rural areas.

Regarding smoking, four patients in each group were smokers. DM and HTN presented in 14 (36.8%) and 7 (18.4%) patients respectively of those with atherosclerosis, 16 (38.3%) and 10 (23.8%) patients respectively of those without atherosclerosis.

The mean body mass index in those patients with atherosclerosis was 26.01 ± 4.90 kg/m2 where 16 (42.1%) of them were normal weight and 14 (36.8%) were overweight respectively while mean body mass index in those patients without atherosclerosis was 28.50 ± 6.42 kg/m2 where 15 (35.7%) of them were normal weight and 10 (23.8%) overweight respectively. As regards the demographic data, all studied groups had no significant differences (P> 0.05).

Clinical Characteristics of Studied Patients:

Table 2 shows the clinical characteristic of studied patients. The mean duration of RA in those with patients with atherosclerosis was 14.08 ± 2.34 years and it was 11.10 ± 2.11 years in those patients without atherosclerosis. The onset of pain was chronic in majority of cases in both groups (in 65.8% of those with atherosclerosis and in 76.2% of those without atherosclerosis).

30 (78.9%) patients with atherosclerosis had morning stiffness with mean duration was 1.10 ± 0.66 hours while morning stiffness presented in 22 (52.4%) of patient without atherosclerosis with mean duration was 1.13 ± 0.58 hours. as regarding the swelling, patients with atherosclerosis had significantly higher feet swelling in comparison to those without atherosclerosis (19 (50%) vs. 10 (23.8%); P 0.03).

There were no significant differences between the studied patients as regarding the extra-articular manifestation.

As regarding systolic blood pressure and diastolic blood pressure, both groups had no significant differences. It was noticed that majority (39.5%) of studied group with atherosclerosis had stage II HTN while 76.2% of those without atherosclerosis had normal blood pressure.

Baseline Laboratory Data of Studied Groups:

All studied groups had no significant differences as regarding baseline laboratory data with exception of:

• Patients with atherosclerosis had significantly higher random blood sugar and higher CRP in comparison to those without atherosclerosis. ESR and HsCRP were significantly higher in both groups of RA.
• Patients with atherosclerosis had significantly higher LDL,, TG and cholesterol and lower HDL in comparison to those without atherosclerosis.
• Patients with atherosclerosis had significantly higher RF in comparison to those without atherosclerosis.(276.76 ± 45.67 vs. 217.91 ± 59.54; P= 0.03).
• It was noticed that level of Anti-CCP was higher in those patients with atherosclerosis in comparison to those without atherosclerosis but with no statistical significance (P= 0.06). Also, majority of studied patients either with or without atherosclerosis had positive Anti-CCP.

Therapeutic History in both Studied Patients:

The therapeutic history of studied patients was summarized at Table 6. Both groups had no significant differences as regarding types of drugs, duration of use and daily dose with exception duration of steroids and Salazopyrin were significantly higher in those with atherosclerosis.

Disease Activity in Studied Patients:

It was noticed that majority (52.6%) of patients with atherosclerosis had high disease activity while 10 (26.3%) and 8 (21.1%) of them had moderate and low disease activity. 5 (11.9%), 17 (40.5%) and 15 (35.7%) of patients without atherosclerosis had low, moderate and high disease activity respectively and 5 (11.9%) were on remission.

As regarding VAS (patients and doctor), and DAS (28ESR and 28CRP), both groups had no significant differences but patients without atherosclerosis had significantly lower MHAQ- DI in comparison to those with atherosclerosis (1.76 ± 0.43 vs. 1.92 ± 0.26; P= 0.04).

OPG titer and OPG (TNFRSF11B) genotypes in Studied Patients:

As regards OPG titer, RA patients with atherosclerosis had higher OPG levels in comparison to those without atherosclerosis (664.34 ± 74.56 vs. 633.34 ± 55.89; P= 0.03).

As regarding types of OPG genotypes, 15 (39.5%), 19 (50%), and 4 (10.5%) of patients with atherosclerosis had CC, CG, and GG genotypes respectively while 18 (42.9%), 20 (47.6%), and 4 (9.5%) of those without atherosclerosis had CC, CG, and GG mutations respectively. It was noticed that the two studied groups had no significant differences as regarding type of OPG genotypes. Also, the C allele was the most frequent allele in all groups.

Types of OPG genotypes and its Titre based on Carotid Atheroma:

It was noticed that OPG’s titre was significantly higher in patients with rheumatoid arthritis who had carotid atheroma in comparison to those without atheroma (684.45 ± 81.53 vs. 641.96 ± 106.65).

Demographic, Clinical, and Laboratory Data of Patients Based on Types of OPG’s Polymorphism:

Table 13 shows the demographic, clinical and laboratory data of studied patients base on OPG’s polymorphism. It was noticed that CC, CG and GG genotypes had no significant differences as regarding demographic, clinical and laboratory data with exception of:

• Patients with GG mutation had significant lower frequency of deformity
• ·RBS and CRP were significantly higher in those patients with CC mutation

Disease Activity, Anti-CCP, RF and OPG’s Titre in Studied Patients Based on OPG’s Polymorphism:

It was noticed that all genotypes of OPG in the current study had no significant differences as regarding disease activity, Anti- CCP, RF, OPG’s titre and sonographic findings with exception of patients with CC and CG mutations had significantly higher Anti- CCP in comparison t those with GG mutation.

Correlation between OPG’sTitre with Different Parameters in the Study:

It was noticed that OPG had insignificant correlation with different parameters in the current study with exception of positive significant correlation with intimal thickness (r=0.37; P= 0.01), RF (r=0.30; P= 0.03) and ACCP (r=0.33; P= 0.03).

Discussion

In our study we had two groups of patients

1. Group I: RA patients with atherosclerosis (no=38).
2. Group II: RA patients without atherosclerosis (no=42),.

All patients with RA were diagnosed and fulfilled American College of Rheumatology /European league against rheumatism (ACR/EULAR) 2010 criteria for RA

To the best of our knowledge, this is the first study to investigate the osteoprotegerin gene polymorphism rs2073618 as a potential marker of subclinical carotid atherosclerosis and RA in Egyptian population.

There were no statistically significant differences between rs2073618 GG, GC, CC genotypes nor G, C alleles among RA patients with atherosclerosis and those without atherosclerosis.

Serum OPG levels were significantly higher in rheumatoid arthritis patients with atherosclerosis (664.34 ± 74.56 ng/L) than patients without atherosclerosis (633.34 ± 55.89 ng/L) (P = 0.03). Also, serum OPG levels were significantly higher in patients with rheumatoid arthritis who had carotid atheroma in comparison to those who didn’t have an atheroma (684.45 ± 81.53 ng/L vs. 641.96 ng/L ± 106.65, P = 0.01).

However, no statistically significant difference could be detected upon comparing median values of serum OPG levels among studied genotype groups (neither genotype nor alleles). The GG genotype had a median level of serum OPG 646.37 ± 85 ng/L, the GC genotype had a median level of serum OPG 668.19 ± 93.02 ng/L, and the CC genotype had a median level of serum OPG 661.09 ± 54.89 ng/L. C allele had a median level of serum OPG 661.11 ± 54.11 ng/L, and the G allele had a median level of serum OPG 665.19 ± 76.89.

Serum OPG levels showed significant positive correlation with RF (r = 0.30, p = 0.03), ACPA (r = 0.33, p = 0.03) and CIMT (r = 0.37, p = 0.01).

Lopez-Mejias et al., 2015 enrolled 151 white Spanish patients who met the 1987 American College of Rheumatology and the 2010 American College of Rheumatology/European League against Rheumatism criteria for RA diagnosis. The aforementioned patients included 54 consecutive patients with established CVD and 97 age-matched and sex-matched cases without CVD. OPG concentrations were also determined in 62 control subjects without CVD.

In agreement with our results; their study showed that RA severity markers, including RF and anti-CCP positivity and erosive disease, were associated with high OPG concentrations. In patients with RA, age, body mass index (BMI), rheumatoid factor (RF) positivity status were significantly correlated with OPG concentrations [partial R (p) = 0.175 (0.03), –0.277 (0.0009), 0.323 (< 0.0001), 0.217 (0.008), and 0.159 (0.05), respectively].

OPG concentrations increased from 6.38 (3.46–9.31) to 7.07 (5.04–10.65) and 8.64 (6.00–11.52) ng/ml {Median (interquartile range)} in controls and RA patients without CVD and those who had CVD, respectively (p = 0.0002). Upon adjustment for age, sex, traditional risk factors, and BMI in mixed regression models, OPG concentrations remained lower in controls compared to patients with RA without CVD (p = 0.05) and in the latter compared to those with CVD (p = 0.03); the association of OPG concentrations with CVD among patients with RA also persisted after additional adjustment for RF and anti-CCP antibody positivity, and erosion status (p = 0.04) (28).

Morisawa et al., 2015 investigated 114 CAD patients (89 men, 25 women; with mean age of 68.7± 10.3 years) and measured the Gensini score (a marker of the extent of coronary atherosclerosis), the mean CIMT, and the plasma levels of OPG and asymmetric dimethylarginine (ADMA; as a marker of endothelial function).

In concordance with our results, Patients with early carotid atherosclerosis had higher OPG levels than those without. The OPG levels were found to be significantly associated with ADMA (r = 0.191, P = 0.046) and the mean CIMT (r = 0.319, P = 0.001), but not with the Gensini score(29).

Pleskovič et al., 2017 investigated whether polymorphism rs2073618 of the OPG gene is associated with subclinical markers of carotid atherosclerosis in subjects with type 2 diabetes mellitus; no statistically significant difference could be detected (p = 0.68) upon comparing median values of serum OPG levels among studied genotype groups when only subjects with type 2 DM were included in the analysis. The GG genotype had a median level of 73.26 (47.03–96) pg/ml, the GC genotype had a median level of 67.56 (44.26–90.43), and the CC genotype had a median level of 61.6 (47.63 -137.89) pg/ml which agrees with our results.

Also in the previous forementioned study; there was no significant difference among CC, CG, and GG genotypes nor C and G allele as regards assessment of CIMT and presence or absence of plaque.(30).

In a study by Soufi et al., 2004 a total of seven different OPG gene polymorphisms were identified in cohort of 468 male patients with or without CAD. Although none of these polymorphisms was associated with the presence of CAD separately which agree with our results, linkage of the 950 and 1181 polymorphisms exhibited a significantly different distribution in patients with CAD compared with patients without CAD. Genotypes 950 TC/1181 GC and 950 CC/1181 CC were overrepresented in men with CAD and were associated with an increased risk of CAD, suggesting that these genotypes may contribute to an increased susceptibility of atherosclerosis (13).

Chung et al., 2015 examined the association between selected genetic polymorphisms and coronary atherosclerosis in patients with RA. Genotypes for single-nucleotide polymorphisms (SNPs) in 152 candidate genes linked with autoimmune or cardiovascular risk were measured in 140 patients with RA. The association between the presence of coronary artery calcium (CAC) and SNP allele frequency was assessed by logistic regression with adjustment for age, sex, and race. 139 patients in whom rs2073618 genotypes were available, 37 (26.6%) had the CC, 62 (44.6%) the CG, and 40 (28.8%) the GG genotypes. Among RA patients with CC genotype, 75.7% had coronary calcium; as compared with 43.6% in those with CG genotype and with 37.5% in those with GG genotype (p = 0.001).

However, in a posthoc analysis examining the association of (rs2073618) genotypes and serum osteoprotegerin concentrations, differences were non-significant (18). Median (interquartile range, IQR) concentrations were 1548 (1042–2509), 1497 (1100–1698), and 1657 (1132–2105) pg/mL, respectively, (p = 0.38).

Genre et al., 2014 examined the role of three functional polymorphisms (rs3134063, rs2073618, and rs3134069) located in the gene encoding osteoprotegerin. The study showed a protective effect of the osteoprotegerin (OPG) CGA haplotype on the risk of cardiovascular disease in patients who were anti-CCP negative. Further examination of the role of individual SNPs suggested that the GG genotype in rs2073618 was associated with a significant reduction of cerebrovascular, but not cardiovascular events (31).

One study showed that the same polymorphism in the gene encoding osteoprotegerin, rs2073618, was associated with atherosclerosis in diabetic patients. The same CC genotype was associated with a three-fold increased risk of stroke in patients with diabetes (15) and the frequency was two-fold higher in patients who underwent carotid endarterectomy compared to control subjects. This last association was even stronger when the results were adjusted for age, sex, hypertension, hypercholesterolemia, diabetes, coronary artery disease, peripheral artery disease, and smoking (32).

Conclusion:

Limitations:

As our study sample size was relatively small, it could produce false-negative results, or it might underestimate the magnitude of the association. Our findings need to be evaluated in larger groups of different ethnicities. In an attempt to clarify the potential role of the OPG/RANKL/ RANK system in atherogenesis, it would have been beneficial to measure not only OPG but also RANKL and RANK serum levels. Interactions between genes of the OPG/RANKL/RANK axis and the development of carotid atherosclerosis remain to be investigated.