Influence of MTHFR C677T gene polymorphism in the development of cardiovascular disease in Egyptian patients with rheumatoid arthritis

Gene 610 (2017) 127–132

Contents lists available at ScienceDirect

Gene journal homepage: www.elsevier.com/locate/gene

Research paper

Influence of MTHFR C677T gene polymorphism in the development of cardiovascular disease in Egyptian patients with rheumatoid arthritis Tarek A. Abd El-Aziz a, Rasha H. Mohamed b,⁎ a b

Cardiology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt Biochemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt

a r t i c l e

i n f o

Article history: Received 15 October 2016 Received in revised form 1 February 2017 Accepted 9 February 2017 Available online 12 February 2017 Keywords: Carotid intima-media thickness Gene polymorphism MTHFR PCR–RFLP RA

a b s t r a c t Objective: To investigate the association between increased carotid intima-media thickness (CIMT), homocysteine level, and MTHFR C677T (rs1801133) gene polymorphism in Egyptian people with rheumatoid arthritis (RA). Subjects and methods: 280 Egyptian women (160 RA patients and 120 controls) were included in the study. CIMT was measured using high resolution B-mode ultrasonography and homocysteine levels were measured using enzyme-linked immunosorbent assay. While, MTHFR C677T polymorphism was analyzed by polymerase chain reaction–restriction fragment length polymorphism. Results: We found that subjects who carried the TT genotype and T allele were significantly more likely to develop RA with 2.9 and 1.5 fold, respectively. RA patients carrying the T allele presented a statistically significant increased risk of developing atherosclerosis compared with those carrying the C allele. Moreover, MTHFR TT genotype was independent risk factor of thick CIMT. Conclusions: C677T MTHFR gene polymorphism is associated with RA in Egyptians. MTHFR 677TT carriers had higher concentrations of serum Hcy than did subjects harboring the CC and CT genotypes. The presence of 677T allele increases the risk of atherosclerosis in patients with RA. This increased risk of atherosclerosis could be due to hyperhomocysteinemia. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Patients with rheumatoid arthritis (RA) have increased risk of cardiovascular disease (CVD) due to accelerated premature atherosclerosis (Gonzalez-Gay et al., 2005). Carotid intima-media thickness (CIMT) reflects early atherosclerosis and predicts cardiovascular events in the general population. An increased CIMT is present in patients with RA, compared with control individuals and is thought to indicate accelerated atherosclerosis (Zanten and Kitas, 2008). It is conceivable that carotid intima-media thickness, which is a well documented surrogate estimate of atherosclerosis and its progression, could become a marker for the early therapeutic intervention to prevent atherosclerosis and cardiovascular disease.

Abbreviation: ANOVA, analysis of variance; CIs, confidence intervals; CIMT, carotid intima-media thickness; CVD, cardiovascular disease; CRP, C-reactive protein; DAS, Disease Activity Score; BMI, body mass index; ELISA, high-sensitivity enzyme-linked immunosorbent assay; ESR, erythrocyte sedimentation rate; MTHFR, methylene tetrahydrofolate reductase; ORs, odds ratios; PCR–RFLP, polymerase chain reaction– restriction fragment length polymorphism; RA, rheumatoid arthritis; RF, rheumatoid factor; TC, total cholesterol; tHcy, total serum homocysteine; TG, triglycerides. ⁎ Corresponding author at: 28-El-Galaa Street, Zagazig 44511, Egypt. E-mail address: [email protected] (R.H. Mohamed).

http://dx.doi.org/10.1016/j.gene.2017.02.015 0378-1119/© 2017 Elsevier B.V. All rights reserved.

Besides classic cardiovascular risk factors, a number of nontraditional cardiovascular risk factors have also been implicated in the elevated cardiovascular mortality observed in these patients (Dessein et al., 2005). In this regard, chronic inflammation and the genetic background increase the risk of cardiovascular events in RA regardless of the presence of traditional cardiovascular risk factors (Gonzalez-Gay et al., 2007). Hyperhomocysteinemia has been found to be an independent nontraditional risk factor for CVD, including coronary disease, in the general population (Clarke et al., 1991). Homocysteine is an intermediary amino acid formed during the conversion of methionine to cysteine. High elevations may be seen in uncommon autosomal defects of the metabolizing enzymes cystathionine β-synthase and 5,10-methylene tetrahydrofolate reductase (MTHFR) (Haagsma et al., 1999). Less severe elevations of homocysteine levels are more commonly observed as a result of heterozygous mutations of these enzymes, dietary deficits of folate or vitamin B12, methotrexate toxicity (Morgan et al., 1998) or in patients with liver disease or decreased renal function (Jacques et al., 2001). Homocysteine is directly toxic to endothelial cells, increases low-density lipoprotein oxidation, and has prothrombotic effects. Increased levels of homocysteine have been found in patients with RA (Hernanz et al., 1999). 5,10-Methylenetetrahydrofolate reductase gene is located on chromosome 1 at 1p36.3. MTHFR enzyme catalyzes irreversible

128

T.A. Abd El-Aziz, R.H. Mohamed Gene 610 (2017) 127–132

reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate which acts as methyl donor for methionine synthesis from homocysteine (Goyette et al., 1998). Many researchers have already tried to find an association between MTHFR polymorphism, homocysteine level, and increased CIMT, but the result remained controversy. Based on this phenomenon, we aimed to investigate the association between MTHFR C677T (rs1801133) gene polymorphism, homocysteine level, and increased CIMT in Egyptian people with RA, and to find the relationship between genetic predisposition and hyperhomocysteinemia as a risk factor of carotid atherosclerosis.

2. Subjects and methods One hundred and sixty female patients with a clinical diagnosis of RA (mean age 34.3 ± 4.2 years) and 120 healthy controls with no past or family history of RA or associated autoimmune disease were enrolled in the study. Characteristics of the RA patients are presented in Table 1. We excluded patients with diabetes (fasting blood sugar N 120 mg), hypertension (systolic blood pressure N 140 mm Hg and/or diastolic blood pressure N 90 mm Hg), dyslipidemia (total cholesterol N 200 mg/dl and triglyceride levels N 150 mg/dl). Obesity (body mass index (BMI) N 30 kg/m2), smoking or previous history of smoking 5 years ago, postmenopausal women, evidence of cardio vascular disease or family history of coronary heart disease. All patients were subjected to history taking especially for presenting symptoms, joint affection, together with history of medications. Disease activity was determined on the basis of defined parameters (the number of swollen and tender joints, Disease Activity Score 28-joint assessment (DAS-28) (Prevoo et al., 1995), C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). X-rays of the hands and feet were obtained in all patients. Disease severity was determined on the basis of defined parameters (titer of rheumatoid factor (RF) and X-ray erosion). Carotid intima-media thickness for RA patients and control was done. The following grading after calculation of the mean of CIMT, CIMT normal if b0.9 mm, while values ≥ 0.9 mm were considered to be indicative of thickened intima. If the maximum CIMT is N1.3 mm, this is an indicative of atherosclerotic plaque (Doria et al., 2003). All Table 1 Characteristic data of RA patients and controls.

Age (years) Disease duration (years) BMI (kg/m2) SBP (mm Hg) DBP (mm Hg) ESR (mm/h) CRP (mg/l) Rheumatoid factor (IU/ml) Total cholesterol (mg/dl) Triglycerides (mg/dl) CIMT ≥ 0.9 mm no (%) Tender joint: no (%) Swollen joint: no (%) DAS 28 Rheumatoid nodule no (%) X-ray erosion: no (%) Rheumatoid factor Positive no (%) Negative no (%) Serum homocysteine (μmol/l) Serum folate (ng/ml)

RA group (n = 160)

Control (n = 120)

P value

34.3 ± 4.2 4.2 ± 2.6 24.9 ± 2.6 116.3 ± 5.8 76.4 ± 4.8 36.6 ± 16.4 5.9 ± 4.0 157.1 ± 63.9 165.1 ± 17.1 78.3 ± 28.2 82 (51.25) 160 (100) 128 (80.0) 4.7 ± 1.1 36 (22.5) 58(36.25) 120 (75.0) 40 (25.0)

33.4 ± 4.5

0.086

24.4 ± 2.5 115.0 ± 5.1 77.5 ± 6.2 33.1 ± 12.2 4.6 ± 1.6 21.1 ± 5.9 141.7 ± 19.3 71.1 ± 14.4 5 (4.2)

0.107 0.052 0.095 0.05 b0.001 b0.001 b0.001 0.011 b0.001

14.3 ± 3.3 16.9 ± 2.9

11.5 ± 1.1 16.2 ± 3.0

b0.001 0.051

Results are expressed as mean ± SD; BMI: body mass index; CIMT: carotid intima-media thickness; CRP: C-reactive protein; DAS: Disease Activity Score; DBP: diastolic blood pressure; ESR: erythrocytes sedimentation rate; SBP: systolic blood pressure.

the patients gave informed consent and the study was approved by the ethical committee of Zagazig University. 2.1. Measurement of carotid intima-media thickness (CIMT) Carotid artery IMT was measured using high resolution B-mode ultrasonography with a L3-11 MHz linear array transducer (Philips Corporation USA). 2.2. Biochemical analysis Blood samples were drawn from all subjects after an overnight fast. Sera were separated immediately and stored at −20 °C. Total cholesterol (Allain et al., 1974) and triglyceride (Siedel et al., 1993) were measured using enzymatic colorimetric assay on Roche/Hitachi Cobas C System (c702) autoanalyzer (Roche Diagnostics, Mannheim, Germany) using CHOL2 and TRIGL reagents, respectively. C-reactive protein (Wu et al., 2002) and RF (Moore and Dorner, 1993) were measured using immunturbidimetric assay on Roche/Hitachi Cobas C System (c501) autoanalyzer (Roche Diagnostics, Mannheim, Germany) using CRPL3 and RFII reagents, respectively. RF was considered positive above 20 IU/ml according to the manufacturer's instructions and a value N 50 IU/ml was considered a high-titer rheumatoid factor according to American College of Rheumatology criteria for rheumatoid arthritis (Aletaha et al., 2010). 2.3. Determination of total serum homocysteine (tHcy) and folic acid Determination of tHcy and folic acid in blood were done by kit uses enzyme-linked immunosorbent assay (Sunred Biological Technology). 2.4. Genomic DNA extraction Genomic DNA was extracted from EDTA whole blood sample using a spin column method according to the protocol (QIAamp Blood Kit; Qiagen GmbH, Hilden, Germany). DNA was stored at − 20 °C till the time of use. 2.5. Amplification of MTHFR C677T (rs1801133) gene polymorphism The subjects were genotyped for MTHFR C677T (rs1801133) polymorphism by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) as described by Frosst et al. (1995). The region surrounding the polymorphism was amplified with the following forward primer 5′-TGAAGGAGAAGGTGTCTGCGGGA-3′ and reverse primer 5′-AGGACGGTGCGTGAGAGTG-3′. PCR was performed at 95 °C for 8 min, followed by 40 cycles at 94 °C for 60 s, at 63 °C for 60 s, and 72 °C for 60 s. A final extension step was carried out at 72 °C for 7 min. After RFLP analysis using enzyme Hinf1 in 37 °C for 1 h, PCR products were separated on 3% agarose stained with ethidium bromide then visualized by ultraviolet light, three genotypes were detected: TT (175, 23 bp), CT (198, 175 and 23 bp) and CC (198 bp). The23 bp fragment was not retained on the gel. 2.6. Statistical analysis The results for continuous variables were expressed as means ± SD. The means of the three-genotype groups were compared in a one-way analysis of variance (ANOVA). Multiple regression analysis was performed to examine the contribution of MTHFR C677T gene polymorphism to carotid IMT. Genotype frequencies in RA cases and controls were tested for Hardy–Weinberg equilibrium, and any deviation between the observed and expected frequencies was tested for significance using the χ2 test. The statistical significances of differences in frequencies of variants between the groups were tested using the chisquare (χ2) test, the student t-test to compare mean values of the 2

T.A. Abd El-Aziz, R.H. Mohamed Gene 610 (2017) 127–132

groups. In addition, the odds ratios (ORs) and 95% confidence intervals (CIs) were calculated as a measure of the association of the MTHFR C677T gene polymorphic site with carotid intima-media thickness. A difference was considered significant at P b 0.05. All data were evaluated using SPSS version 10.0 of windows. 3. Results 3.1. Characteristics of the study population (Table 1) The frequency distributions of selected characteristics of the cases and controls are presented in Table 1. There was no significant difference in the blood pressure, BMI, or triglyceride levels between the cases and controls. Carotid IMT was increased in patients with RA compared with matched controls. ESR, CRP, RF, TC, and serum homocysteine levels were increased in RA patients compared with controls. 3.2. Relation of parameters of RA disease activity and severity with atherosclerosis (Table 2) We found that the RA disease activity (DAS28), disease severity (RF and X-ray erosion) parameters, total cholesterol, and homocysteine levels were significantly higher in RA patients suffering from atherosclerosis than those without atherosclerosis. Moreover, 15.4% of non-atherosclerotic RA patients had rheumatoid nodules meanwhile 29.3% of atherosclerotic RA patients had rheumatoid nodules. 3.3. Distribution of MTHFR C677T genotype and alleles in RA patients and control groups (Table 3) Regarding genotypic distribution, both cases and controls were in Hardy-Weinberg equilibrium for the two polymorphisms. The frequency of the TT genotype of MTHFR was significantly increased in RA group compared to control group (15.0% versus 5.8%). Subjects with the TT genotype were significantly more likely to develop RA (OR = 2.9, 95% CI = 1.2–7.3, P = 0.011). 3.4. Frequencies of gene polymorphisms in RA patients with or without atherosclerosis (Table 4) Table 4 presented the genotype frequencies of the MTHFR C677T gene polymorphism assessed in this study of RA patients stratified by the presence of atherosclerosis. MTHFR 677TT genotype frequency was 19.5% in RA patients with atherosclerosis versus 10.3% in those RA patients without atherosclerosis. Interestingly, RA patients carrying

129

minor T allele were significantly more likely to develop atherosclerosis compared with those carrying the C allele (OR = 4.6, 95% CI = 2.8– 7.8, P ˂ 0.001). 3.5. Distribution of common risk factors for atherosclerosis in RA across genotypes (Table 5) Higher Carotid IMT (1.0 ± 0.2 vs. 0.8 ± 0.09 mm), serum homocysteine (19.1 ± 1.3 vs. 11.9 ± 1.7 μmol/l), TC levels (169.2 ± 20.2 vs. 161.1 ± 14.6 mg/dl), TG levels (104.3 ± 31.1 vs. 72.3 ± 23.3 mg/dl), CRP levels (6.7 ± 4.4 vs. 4.9 ± 2.1 mg/l), and ESR (36.5 ± 23.6 vs. 29.2 ± 15.6 mm/h) were observed in RA patients carrying the 677TT genotype compared with RA patients carrying the 677CC genotype. 3.6. Multiple regression analysis of carotid IMT (Table 6) In the multiple regression model with carotid IMT as the dependent variable, Disease duration of RA, age, BMI, and DAS were independent risk factors of thick carotid IMT. Moreover, MTHFR TT genotype was independent risk factor of thick CIMT. 4. Discussion Large epidemiological studies from the last few decades have confirmed that patients with RA are 60% more likely to suffer a CVD event than subjects from the general population (Meune et al., 2009). The major complication in patients with RA is the development of cardiovascular events due to accelerated atherosclerosis. Multiple studies have confirmed that the excess mortality in RA is largely attributed to CVD death. A meta-analysis of 24 studies showed a 50% increased risk of cardiovascular death overall (Aviña–Zubieta et al., 2008). Accordingly, clinicians should be aware of the high risk and provide close surveillance of CVD in patients with RA. In the general population, carotid ultrasound has been used for cardiovascular risk stratification; intimamedial thickness (IMT) is associated with clinical CVD and has independent prognostic value for CV events (Roman et al., 2006). A common C677T polymorphism in the gene coding for the MTHFR enzyme has been found to be a new candidate genetic risk factor for CV disease in the general population (Xuan et al., 2014; Hmimech et al., 2016). In this study we investigated the association of MTHFR C677T polymorphism with risk of atherosclerosis in RA patients and the effect of this polymorphism on carotid intima media thickness and homocysteine level.

Table 2 Relation of atherosclerosis and parameters of RA disease activity and severity.

Disease duration(years) Swollen joints Tender joints DAS28 ESR (mm/h) CRP (mg/l) Rheumatoid factor (IU/ml) Rheumatoid nodule no (%) X-ray erosion: no (%) Serum homocysteine (μmol/l) Total cholesterol (mg/dl) Triglycerides (mg/dl) SBP (mm Hg) DBP (mm Hg)

RA patients with CIMT b 0.9 mm no = 78

RA patients with CIMT ≥ 0.9 mm no = 82

P

3.2 ± 1.6 2.7 ± 0.6 4.9 ± 1.2 4.5 ± 1.1 38.1 ± 3.4 6.1 ± 4.1 150.3 ± 14.9 12 (15.4) 20 (25.6) 13.2 ± 2.9 167.6 ± 18.1 81.9 ± 31.1 116.7 ± 6.5 75.6 ± 5.0

4.6 ± 3.4 2.8 ± 0.3 8.4 ± 2.1 4.9 ± 0.9 35.3 ± 7.4 5.7 ± 3.2 163.6 ± 50.2 24 (29.3) 38 (46.3) 15.4 ± 3.3 162.2 ± 15.2 74.8 ± 14.0 115.8 ± 4.9 77.1 ± 4.6

0.001 0.181 b0.001 0.012 0.002 0.491 0.026 0.024 0.009 b0.001 0.040 0.062 0.323 0.051

Results are expressed as mean ± SD; CIMT: carotid intima-media thickness; CRP: C-reactive protein; DAS: Disease Activity Score; DBP: diastolic blood pressure; ESR: erythrocytes sedimentation rate; SBP: systolic blood pressure.

130

T.A. Abd El-Aziz, R.H. Mohamed Gene 610 (2017) 127–132

Table 3 Frequency distributions of MTHFR genotypes and alleles.

MTHFR C677T

Genotypes/alleles

RA group (n = 160) n (%)

Control group (n = 120) n (%)

OR (95% CI)

P

TT CT CC T allele C allele

24 (15.0) 57 (35.6) 79 (49.4) 105 (32.8) 215 (67.2)

7 (5.8) 45 (37.5) 68 (56.7) 59 (24.6) 181 (75.4)

2.9 (1.2–7.3) 0.9 (0.55–1.5)

0.011 0.412

1.5 (1.02–2.2)

0.021

MTHFR: methylene tetrahydrofolate reductase; RA: rheumatoid arthritis.

To the best of our knowledge, this is the first study to evaluate the association of C677T MTHFR polymorphism with RA and atherosclerosis in Egyptians. In our study, RA patients had elevated levels of RF, erythrocyte sedimentation rate, and CRP than controls (Table 1) which is in agreement with previous study (Peters et al., 2010). The accelerated atherosclerosis has been reported in RA to be independent of traditional risk factors (Solomon et al., 2004). In the present study, diabetes mellitus, hypertension, obesity and smoking were exclusion criteria while the mean values of total cholesterols and triglycerides were within normal range (Table 1), thus our study was free of the effects of these traditional risk factors for accelerated atherosclerosis. Chung et al. (2005) and Mahajan et al. (2008) showed no significant correlation between dyslipidaemia and accelerated atherosclerosis in RA patients. Also, Jonsson et al. (2001) and Roman et al. (2006) reported the same results. Excess CVD mortality occurs predominantly in RA patients with higher degree of systemic inflammation (Maradit-Kremers et al., 2005). Thus, active RA with high degree of inflammation may lead to accelerated atherosclerosis and increased CVD morbidity and mortality. In our study, we found that the total serum levels of homocysteine were significantly higher in RA patients than in the control group but the serum levels of folate were within the reference range in RA patients (Table 1). These results were in agreement with studies done by (Morgan et al., 1991) and (Kim et al., 1996) who showed that plasma levels of folate, and red cell folate were within the reference range in patients with RA. Furthermore, a study done by (Roubenoff et al., 1997) reported that the plasma homocysteine levels significantly increased in RA patients. The present study showed a significantly higher value of CIMT in RA patients than the normal population (i.e., non-invasive evidence of atherosclerosis). A similar observation has also been shown by previous studies (Alkaabi, 2003; Mahajan et al., 2008). In our study, comparing clinical and laboratory features of RA patients with CIMT b 0.9 mm and those with CIMT ≥ 0.9 mm revealed that RA patients with CIMT ≥ 0.9 mm have significant increase in number of tender joints, DAS28, levels of RF, and serum Hcy levels (Table 2). Moreover, RA patients with CIMT ≥ 0.9 mm had significant increase in disease duration (Table 2), suggesting that carotid IMT increased with increased duration of disease. Mahajan et al. (2008) found disease

duration as one of the best predictor for the development of severe morphologic expression of atherosclerotic disease. In the general population, carotid ultrasound has been used for cardiovascular risk stratification; intima-medial thickness and plaque are associated with clinical cardiovascular disease and have independent prognostic value for cardiovascular events (Roman et al., 2006). In study of Asian patients (Park et al., 2002) and in a study from Poland (Surdacki et al., 2007), CIMT was increased in patients with RA compared with matched controls. A meta-analysis was performed involving 22 studies to estimate the overall mean CIMT difference between RA and control groups. In 17 of the studies, patients with RA had a statistically significant increase of carotid IMT (van Sijl et al., 2011). The overall mean CIMT difference was 0.09 mm, indicated an approximately 15% increased cardiovascular risk (Bots et al., 1997; Wu et al., 2013). Bożena et al. (2011) reported that the values of CIMT were significantly greater in RA patients compared to control subjects. A significant relation of features of RA even in the absence of traditional clinical CV risk factors, were associated with greater CIMT in RA patients. As regards MTHFR C677T, we found that the CC genotype was the most frequent among patients (49.4%) compared to the other genetic profiles (35.6% CT and 15% TT). In healthy controls, the CC genotype was the most frequent (56.7%) vs. 37.5% and 5.8% for the CT and TT genetic profiles, respectively. The prevalence of 677T allele was 32.8% in RA patients vs. 24.6% in controls. The prevalence of MTHFR polymorphisms varies in different geographical regions and ethnic groups. The prevalence of 677T allele ranged from 24.1% to 64.3% among Europeans (Gueant-Rodriguez et al., 2006), 2–48% among North Americans, 35.5% among Africans (Rajeevan et al., 2012) and 2% to 63.1% in Asians (Rajeevan et al., 2012). In Arab population, the prevalence of MTHFR C677T mutation is 24% in Jordan (Eid and Rihani, 2004) and 46.9% in Saudi Arabia (Bu et al., 2004). Inanir et al. (2013) reported that there was no statistical significant difference in the genotype frequencies at MTHFR gene in RA, but allele frequencies showed statistically significant association with RA. In the present study, we found that subjects with the 677TT genotype had higher concentrations of serum Hcy (P ˂ 0.001) and great carotid atherosclerosis (higher CIMT). Moreover, increase in disease activity laboratory features of RA (CRP; P = 0.007; and ESR; P = 0.018) and dyslipidemia (P ≤ 0.02).

Table 4 Frequencies of MTHFR C677T gene polymorphism in RA patients with or without atherosclerosis.

MTHFR C677T

Genotypes/alleles

RA patients with CIMT b 0.9 mm (n = 78) n (%)

RA patients with CIMT ≥ 0.9 mm (n = 82) n (%)

OR (95% CI)

P

TT CT CC T C

8 (10.3) 10 (12.8) 60 (76.9) 26 (16.7) 130 (83.3)

16 (19.5) 47 (57.3) 19 (23.2) 79 (48.2) 85 (51.8)

6.3 (2.3–17.1) 14.8 (6.3–34.9)

b0.001 b0.001

4.6 (2.8–7.8)

b0.001

CIMT: carotid intima-media thickness; MTHFR: methylene tetrahydrofolate reductase; RA: rheumatoid arthritis.

T.A. Abd El-Aziz, R.H. Mohamed Gene 610 (2017) 127–132 Table 5 Distribution of common risk factors for atherosclerosis in rheumatoid arthritis across genotypes. C677T polymorphism

CIMT (mm) tHcy (μmol/l) TC (mg/dl) TG (mg/dl) CRP (mg/l) RF (IU/ml) DAS28 ESR (mm/h)

CC N = 79

CT N = 57

P

TT N = 24

P

0.8 ± 0.09 11.9 ± 1.7 161.1 ± 14.6 72.3 ± 23.3 4.9 ± 2.1 156.4 ± 50.1 4.5 ± 1.1 29.2 ± 15.6

0.93 ± 0.1a 15.6 ± 2.6a 166.7 ± 17.1a 75.6 ± 27.3 6.4 ± 4.8a 150.5 ± 74.3 4.8 ± 1.1 42.1 ± 31.8

b0.001 b0.001 0.039 0.450 0.014 0.581 0.119 0.002

1.0 ± 0.2a 19.1 ± 1.3a 169.2 ± 20.2a 104.3 ± 31.1a 6.7 ± 4.4a 175.1 ± 87.3 4.9 ± 1.6 36.5 ± 23.6a

b0.001 b0.001 0.021 b0.001 0.007 0.189 0.213 0.018

Results are expressed as mean ± SD; CIMT: carotid intima-media thickness; CRP: C-reactive protein; DAS: Disease Activity Score; ESR: erythrocytes sedimentation rate; RF: rheumatoid factor; tHcy: total homocysteine; TC: total cholesterol; TG: triglyceride. a Genotypes CC versus CT and TT.

In accordance with our results, Gudnason et al. (1997) reported that 677T allele was found to be associated with higher CIMT in men. This result is consistent with the known association between this allele and elevated homocysteine levels which were also related to higher CIMT and cardiovascular diseases. Arai et al. (1997) found that 677TT genotype is associated with higher CIMT values in non-insulin dependant diabetic subjects. Moreover, Demuth et al. (1998) have shown a negative association between the carotid diameter and T677 allele in subjects with no atherosclerotic lesion. Pallaud et al. (2001) and Pramukarso et al. (2015) reported no association between MTHFR genetic polymorphisms and CIMT progression. In the present study, multiple regression analysis showed that 677TT genotype is an independent risk factor of increased CIMT. However, Hcy was not independent risk factor of increased CIMT, though it was significantly increased in 677TT genotype compared to 677CC genotype in atherosclerotic RA patients. This suggests that an interaction exists between the 677TT genotype and serum Hcy (Table 6). Indeed, it has been suggested that plasma Hcy is a mediator in the linkage between MTHFR genotype and carotid atherosclerosis (Liu et al., 2007). Previous studies have reported that individuals carrying a the 677T allele had significantly increased plasma levels of homocysteine, and suggested that this variant may be a potential genetic risk factor for cardiovascular diseases (Akar et al., 2000; Klerk et al., 2002). In addition, Frosst et al. (1995) suggested a link between genotype mutant homozygotes TT with hyperhomocysteinemia. It has been known that a T allele carrier at greater risk for hyperhomocysteinemia compared to C allele. On the contrary, Pramukarso et al. (2015) reported that CC alleles associated with the occurrence of hyperhomocysteinemia. Table 6 Multiple regression analysis of carotid IMTa.

Disease duration(years) Age (years) BMI (kg/m2) DAS28 ESR (mm/h) RF (IU/ml) CRP (mg/l) TC (mg/dl) TG (mg/dl) tHcy (μmol/l) MTHFR (TT genotype)

Bb

t

P

0.49 0.41 0.22 0.62 −0.88 0.15 0.12 0.18 0.84 0.18 0.58

4.4125 3.43 2.17 3.30 −0.45 1.94 1.22 1.67 7.14 1.75 4.59

b0.001 0.001 0.034 0.002 0.662 0.05 0.230 0.091 b0.001 0.082 0.013

BMI: Body mass index; CRP: C-reactive protein; DAS: Disease Activity Score; ESR: erythrocytes sedimentation rate; MTHFR: methylene tetrahydrofolate reductase; RF: rheumatoid factor; tHcy: total homocysteine; TC: total cholesterol; TG: triglyceride. a Dependant variable: Carotid IMT. b Standardized coefficient.

131

5. Conclusion C677T MTHFR gene polymorphism is associated with RA in Egyptians. MTHFR 677TT carriers had higher concentrations of serum Hcy than did subjects harboring the CC and CT genotypes. The presence of 677 T allele increases the risk of atherosclerosis in patients with RA. This increased risk of atherosclerosis could be due to hyperhomocysteinemia. However, we are limited by the small sample which might decrease the statistical power of our study; thus, we recommend further larger studies to determine whether MTHFR C677T gene polymorphism is an early marker for occurrence of atherosclerosis in patients with RA. Conflict of interest No conflict of interest. References Allain, C.C., Poon, L.S., Chan, C.S., Richmond, W., Fu, P.C., 1974. Enzymatic determination of total serum cholesterol. Clin. Chem. 20, 470–475. Akar, N., Akar, E., Akçay, R., Avcu, F., Yalcin, A., Cin, S., 2000. Effect of methylenetetrahydrofolate reductase 677 C-T, 1298 A-C, and 1317 T-C on factor V 1691 mutation in Turkish deep vein thrombosis patients. Thromb. Res. 97, 163–167. Alkaabi, K., 2003. Rheumatoid arthritis and macrovascular disease. Rheumatology 42, 292–297. Aletaha, D., Neogi, T., Silman, A., Funovits, J., Felson, D.T., Bingham, C.O., Birnbaum, N.S., Burmester, G.R., Bykerk, V.P., Cohen, M.D., Combe, B., Costenbader, K.H., Dougados, M., Emery, P., Ferraccioli, G., Hazes, J.M.W., Hobbs, K., Huizinga, T.W.J., Kavanaugh, A., Kay, J., Kvien, T.K., Laing, T., Mease, P., Ménard, H.A., Moreland, L.W., Naden, R.L., Pincus, T., Smolen, J.S., Stanislawska-Biernat, E., Symmons, D., Tak, P.P., Upchurch, K.S., Vencovský, J., Wolfe, F., Hawker, G., 2010. Rheumatoid arthritis classification criteria, an American college of rheumatology/European league against rheumatism collaborative initiative. Ann. Rheum. Dis. 69, 1580–1588. Arai, K., Yamasaki, Y., Kajimoto, Y., Watada, H., Umayahara, Y., Kodama, M., Sakamoto, K., Hori, M., 1997. Association of methylenetetrahydrofolate reductase gene polymorphism with carotid arterial wall thickening and myocardial in fraction risk in NIDDM. Diabetes 46, 2102–2104. Aviña–Zubieta, J.A., Choi, H.K., Sadatsafavi, M., Etminan, M., Esdaile, J.M., Lacaille, D., 2008. Risk of cardiovascular mortality in patients with rheumatoid arthritis: a meta-analysis of observational studies. Arthritis Rheum. 59, 1690–1697. Bots, M.L., Hoes, A.W., Koudstaal, P.J., Hofman, A., Grobbee, D.E., 1997. Common carotid intima-media thickness and risk of stroke and myocardial infarction: the Rotterdam study. Circulation 96, 1432–1437. Bożena, T.S., Anna, D.Z., Maria, M., 2011. The relationship between carotid intima-media thickness and the activity of rheumatoid arthritis. J. Clin. Rheumatol. 17, 249–255. Bu, R., Gutierrez, M.I., Al-Rasheed, M., Belgaumi, A., Bhatia, K., 2004. Variable drug metabolism genes in Arab population. Pharmacogenomics J. 4, 260–266. Clarke, R., Daly, L., Robinson, K., Naughten, E., Cahalane, S., Fowler, B., Graham, I., 1991. Hyperhomocysteinemia: an independent risk factor for vascular disease. N. Engl. J. Med. 324, 1149–1155. Chung, C.P., Oeser, A., Raggi, P., Gebretsadik, T., Shintani, A.K., Sokka, T., Pincus, T., Avalos, I., Stein, C.M., 2005. Increased coronary artery atherosclerosis in rheumatoid arthritis. Am. Col. Rheumatol. 52, 2045–2053. Demuth, K., Moatti, N., Hanon, O., Benoit, M.O., Safar, M., Girerd, X., 1998. Opposite effects of plasma homocysteine and the methylenetetrahydrofolate reductase C677T mutation on carotid artery geometry in asymptomatic adults. Arterioscler. Thromb. Vasc. Biol. 18, 1838–1843. Dessein, P.H., Joffe, B.I., Veller, M.G., Stevens, B.A., Tobias, M., Reddi, K., Stanwix, A.E., 2005. Traditional and nontraditional cardiovascular risk factors are associated with atherosclerosis in rheumatoid arthritis. J. Rheumatol. 32, 435–442. Doria, A., Shoenfeld, A., Wu, R., Gambari, P.F., Puato, M., Ghirardello, A., Gilburd, B., Corbanese, S., Patnaik, M., Zampieri, S., Peter, J.B., Favaretto, E., Iaccarino, L., Sherer, Y., Todesco, S., Pauletto, P., 2003. Risk factors for subclinical atherosclerosis in a prospective cohort of patients with systemic lupus erythematosus. Ann. Rheum. Dis. 62, 1071–1077. Eid, S.S., Rihani, G., 2004. Prevalence of factor V Leiden, prothrombin G20210A, and MTHFR C677T mutations in 200 healthy Jordanians. Clin. Lab. Sci. 17, 200–202. Frosst, P., Blom, H.J., Milos, R., Goyette, P., Sheppard, C.A., Matthews, R.G., Boers, G.J., den Heijer, M., Kluijtmans, L.A., van den Heuvel, L.P., 1995. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat. Genet. 10, 111–113. Gonzalez-Gay, M.A., Gonzalez-Juanatey, C., Martin, J., 2005. Rheumatoid arthritis: a disease associated with accelerated atherogenesis. Semin. Arthritis Rheum. 35, 8–17. Gonzalez-Gay, M.A., Gonzalez-Juanatey, C., Lopez-Diaz, M.J., Piñeiro, A., Garcia-Porrua, C., Miranda-Filloy, J.A., Ollier, W.E., Martin, J., Llorca, J., 2007. HLA-DRB1 and persistent chronic inflammation contribute to cardiovascular events and cardiovascular mortality in patients with rheumatoid arthritis. Arthritis Rheum. 57, 125–132. Goyette, P., Pai, A., Milos, R., Frosst, P., Tran, P., 1998. Gene structure of human and mouse methylene-tetrahydrofolate reductase (MTHFR). Mamm. Genome 9, 652–656.

132

T.A. Abd El-Aziz, R.H. Mohamed Gene 610 (2017) 127–132

Gueant-Rodriguez, R.M., Guéant, J., Debard, R., Thirion, S., Hong, L.X., Bronowicki, J.P., Namour, F., Chabi, N.W., Sanni, A., Anello, G., Bosco, P., Romano, C., Amouzou, E., Arrieta, H.R., Sánchez, B.E., Romano, A., Herbeth, B., Guilland, J.C., Mutchinick, O.M., 2006. Prevalence of methylenetetrahydrofolate reductase 677T and 1298C alleles and folate status: a comparative study in Mexican, West African, and European populations. Am. J. Clin. Nutr. 83, 701–707. Gudnason, V., Thormar, K., Humphries, S.E., 1997. Interaction of the cholesteryl ester transfer protein I405V polymorphism with alcohol consumption in smoking and non-smoking healthy men, and the effect on plasma HDL cholesterol and apoAI concentration. Clin. Genet. 51, 15–21. Haagsma, C.J., Blom, H.J., van Riel, P.L., van't Hof, M.A., Giesendorf, B.A., van OppenraaijEmmerzaal, D., Putte, L.B., 1999. Influence of sulphasalazine, methotrexate, and the combination of both on plasma homocysteine concentrations in patients with rheumatoid arthritis. Ann. Rheum. Dis. 58, 79–84. Hernanz, A., Plaza, A., Martin-Mola, E., De Miguel, E., 1999. Increased plasma levels of homocysteine and other thiol compounds in rheumatoid arthritis women. Clin. Biochem. 32, 65–70. Hmimech, W., Idrissi, H.H., Diakite, B., Baghdadi, D., Korchi, F., Habbal, R., Nadifi, S., 2016. Association of C677T MTHFR and G20210A FII prothrombin polymorphisms with susceptibility to myocardial infarction. Biomedic. Rep. 5, 361–366. Inanir, A., Yigit, S., Tekcan, A., Tura, S., Kismali, G., 2013. IL-4 and MTHFR gene polymorphism in rheumatoid arthritis and their effect. Immunol. Lett. 152, 104–108. Jacques, P.F., Bostom, A.G., Wilson, P.W., Rich, S., Rosenberg, I.H., Selhub, J., 2001. Determinants of plasma total homocysteine concentration in the Framingham offspring cohort. Am. J. Clin. Nutr. 73, 613–621. Jonsson, S.W., Backman, C., Johnson, O., Karp, K., Lundström, E., Sundqvist, K.G., Dahlqvist, S.R., 2001. Increased prevalence of atherosclerosis in patients with medium term RA. J. Rheumatol. 28, 2597–2602. Kim, Y.I., Logan, J.W., Mason, J.B., Roubenoff, R., 1996. DNA hypomethylation in inflammatory arthritis: reversal with methotrexate. J. Lab. Clin. Med. 128, 165–172. Klerk, M., Verhoef, P., Clarke, R., Blom, H.J., KOK, F.J., Schouten, E.G., 2002. MTHFR 677C N T polymorphism and risk of coronary heart disease: a meta-analysis. JAMA 288, 2023–2031. Liu, C.S., Chen, C.H., Chiang, H.C., Kuo, C.L., Huang, C.S., Cheng, W.L., Wei, Y.H., Chen, H.W., 2007. B-group vitamins, MTHFR C677T polymorphism and carotid intima-media thickness in clinically healthy subjects. Eur. J. Clin. Nutr. 61, 996–1003. Mahajan, V., Handa, R., Kumar, U., Sharma, S., Gulati, G., Pandey, R.M., Lakshmy, R., 2008. Assessment of atherosclerosis by carotid intimomedial thickness in patients with rheumatoid arthritis. JAPI 56, 587–590. Maradit-Kremers, H., Nicola, P.J., Crowson, C.S., Ballman, K.V., Gabriel, S.E., 2005. Cardiovascular death in rheumatoid arthritis: a population-based study. Arthritis Rheum. 52, 722–732. Meune, C., Touzé, E., Trinquart, L., Allanore, Y., 2009. Trends in cardiovascular mortality in patients with rheumatoid arthritis over 50 years: a systematic review and meta-analysis of cohort studies. Rheumatology (Oxford) 48, 1309–1313. Moore, T.L., Dorner, R.N., 1993. Rheumatoid factors. Clin. Biochem. 26, 75–84. Morgan, S.L., Baggott, J.E., Refsum, H., Ueland, P.M., 1991. Homocysteine levels in patients with rheumatoid arthritis treated with low-dose methotrexate. Clin. Pharmacol. Ther. 50, 547–556. Morgan, S.L., Baggott, J.E., Lee, J.Y., Alarcon, G.S., 1998. Folic acid supplementation prevents deficient blood folate levels and hyperhomocysteinemia during long term, low dose methotrexate therapy for rheumatoid arthritis: implications for cardiovascular disease prevention. J. Rheumatol. 25, 441–446. Pallaud, C., Sass, C., Zannad, F., Siest, G., Visvikis, S., 2001. APOC3, CETP, fibrinogen, and MTHFR are genetic determinants of carotid intima-media thickness in healthy men (the Stanislas cohort). Clin. Genet. 59, 316–324.

Park, Y.B., Ahn, C.W., Choi, H.K., Lee, S.H., In, B.H., Lee, H.C., Nam, C.M., Lee, S.K., 2002. Atherosclerosis in rheumatoid arthritis: morphologic evidence obtained by carotid ultrasound. Arthritis Rheum. 46, 1714–1719. Peters, M.J., Symmons, D.P., McCarey, D., Dijkmans, B.A., Nicola, P., Kvien, T.K., McInnes, I.B., Haentzschel, H., Gonzalez-Gay, M.A., Provan, S., Semb, A., Sidiropoulos, P., Kitas, G., Smulders, Y.M., Soubrier, M., Szekanecz, Z., Sattar, N., Nurmohamed, M.T., 2010. EULAR evidence-based recommendations for cardiovascular risk management in patients with rheumatoid arthritis and other forms of inflammatory arthritis. Ann. Rheum. Dis. 69, 325–331. Pramukarso, D.T., Faradz, S.M.H., Sari, S., Hadisaputro, S., 2015. Association between methylenetetrahydrofolate reductase (MTHFR) polymorphism and carotid intima medial thickness progression in post ischaemic stroke patient. Ann. Transl. Med. 324, 1–5. Prevoo, M.L., van't Hof, M.A., Kuper, H.H., van Leeuwen, M.A., vande Putte, L.B., van Riel, P.L., 1995. Modified disease activity scores that include twenty-eight-joint counts: development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum. 38, 44–48. Rajeevan, H., Soundararajan, U., Pakstis, A.J., Kidd, K.K., 2012. Introducing the forensic research/reference on genetics knowledge base, FROG-kb. Investig. Genet. 3, 1–8. Roman, M.J., Naqvi, T.Z., Gardin, J.M., Gerhard-Herman, M., Jaff, M., Mohler, E., American Society of Echocardiography, Society of Vascular Medicine and Biology, 2006. Clinical application of noninvasive vascular ultrasound in cardiovascular risk stratification: a report from the American society of echocardiography and the society of vascular medicine and biology. J. Am. Soc. Echocardiogr. 19, 943–954. Roubenoff, R., Dellaripa, P., Nadeau, M.R., Abad, L.W., Muldoon, B.A., Selhub, J., Rosenberg, I.H., 1997. Abnormally homocysteine metabolism in rheumatoid arthritis. Arthritis Rheum. 40, 718–722. Siedel, J., Schmuck, R., Staepels, J., 1993. Long term stable, liquid ready-to-use monoreagent for the enzymatic assay of serum or plasma triglycerides (GPO-PAP method). AACC Meeting Abstract 34. Clin. Chem. 39, 1127. Solomon, D.H., Schneeweiss, S., Glynn, R.J., Kiyota, Y., Levin, R., Mogun, H., Avorn, J., 2004. Relationship between selective cyclooxygenase-2 inhibitors and acute myocar-dial infarction in older adults. Circulation 109, 2068–2073. Surdacki, A., Martens-Lobenhoffer, J., Wloch, A., Marewicz, E., Rakowski, T., WieczorekSurdacka, E., Dubiel, J.S., Pryjma, J., Bode-Boger, S.M., 2007. Elevated plasma asymmetric dimethyl 1-1 arginine levels are linked to endothelial progenitor cell depletion and carotid atherosclerosis in rheumatoid arthiritis. Arthritis Rheum. 56, 809–819. van Sijl, A.M., Peters, M.J., Knol, D.K., de Vet, H.C., Gonzalez-Gay, M.A., Smulders, Y.M., Dijkmans, B.A., Nurmohamed, M.T., 2011. Carotid intima media thickness in rheumatoid arthritis as compared to control subjects: a meta-analysis. Semin. Arthritis Rheum. 40, 389–397. Wu, T.L., Tsao, K.C., Chang, C.P., Li, C.N., Sun, C.F., Wu, J.T., 2002. Development of ELISA on microplate for serum C-reactive protein and establishment of age-dependent normal reference range. Clin. Chim. Acta 322, 163–168. Wu, M., Zeng, F.F., Wang, R., Seto, W.K., Pai, P., Chu, P., Yiu, K.H., 2013. Atherosclerosis in patients with rheumatoid arthritis. Rheumatol. Curr. Res. S5. Xuan, C., Li, H., Zhao, J.X., Wang, H.W., Wang, Y., Ning, C.P., Liu, Z., Zhang, B.B., He, G.W., Lun, L.M., 2014. Association between MTHFR polymorphisms and congenital heart disease: a meta-analysis based on 9,329 cases and 15,076 controls. Sci. Rep. 4, 7311. Zanten, V.J.J., Kitas, G.D., 2008. Inflammation, carotid intima-media thickness and atherosclerosis in rheumatoid arthritis. Arthritis. Res. Ther. 10, 102.