Clinical significance of TNFAIP3 rs2230926 T > G gene polymorphism in Egyptian cases with rheumatoid arthritis

Meta Gene 11 (2017) 58–63

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Clinical significance of TNFAIP3 rs2230926 T N G gene polymorphism in Egyptian cases with rheumatoid arthritis Samy Y. Elkhawaga a,⁎, Ahmed I. Abulsoud a, Mostafa M. Elshafey a, Mohsen M. Elsayed b a b

Biochemistry Department, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo, Egypt Rheumatology and Rehabilitation Department, Faculty of Medicine (Boys), Al-Azhar University, Cairo, Egypt

a r t i c l e

i n f o

Article history: Received 9 September 2016 Revised 18 October 2016 Accepted 10 November 2016 Available online 15 November 2016 Keywords: TNFAIP3 A20 Gene polymorphism Rheumatoid arthritis Egyptian

a b s t r a c t Background: Rheumatoid arthritis (RA) is an inflammatory autoimmune disease. The tumor necrosis factor alphainduced protein 3 (TNFAIP3), which encodes ubiquitin-editing protein A20 has been reported to be associated with RA in some populations. We aimed to assess the association of TNFAIP3 rs2230926 TN G gene polymorphism with the susceptibility, activity and functional disability of RA in Egyptian patients. Subjects and methods: This study included 82 unrelated RA patients and 81 unrelated healthy individuals from the same region. DNA of all subjects was genotyped for TNFAIP3 rs2230926 TN G polymorphism by Real Time-PCR using TaqMan® allele discrimination assay. Results: RA patients showed significantly higher TNFAIP3 rs2230926 G allele carriage (TG + GG genotypes) compared to controls (35.37% vs. 14.82%, OR = 3.15, 95% CI = 1.47–6.74, P = 0.0036). Also, the frequencies of the rs2230926 G allele were significantly higher among patients compared to controls (18.30% vs. 7.40%, OR = 2.8, 95% CI = 1.38–5.69, P = 0.0045). Patients carrying TG + GG genotypes showed no significant differences from those with TT genotype regarding clinical and laboratory findings except for the rheumatoid deformity (P = 0.030) and health assessment questionnaire (HAQ) score (P = 0.004) that was significantly higher between patients carrying G allele. Furthermore, TNFAIP3 rs2230926 G allele had significantly improved the risk to develop joint deformity in seronegative RA patients for rheumatoid factor (RF) or anti-cyclic citrullinated peptide (Anti-CCP) antibody (OR = 19, 95% CI = 1.65–218.6, P = 0.015, OR = 6.33, 95% CI = 1.20–33.4, P = 0.04 respectively). Conclusions: TNFAIP3 rs2230926 G allele is associated with susceptibility to RA, and functional disability in patients with rheumatoid arthritis. This may provide a good marker for the diagnosis of RA susceptibility and help in the prediction of disease severity. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Rheumatoid arthritis (RA) is the most common inflammatory autoimmune disease characterized by synovial joints inflammation leading to joint destruction and severe disability, affecting up to 1% of adults population (Firestein, 2003). Although the etiology of RA remains not understood, both genetic component and environmental factors play a central role in disease development. Genetic factors were considered to be liable for up to 60% of predisposition to RA (MacGregor et al., 2000; Salama et al., 2014). Rheumatoid arthritis is characterized by the presence of different autoantibodies (Gourraud et al., 2006). Best well-known is the rheumatoid factor (RF), which is an antibody against the Fc part of the immunoglobulin G molecules, is present in 70% of RA patients and the detection of RF (IgM) is used as serological marker in diagnosis of RA; high level of ⁎ Corresponding author. E-mail address: [email protected] (S.Y. Elkhawaga).

http://dx.doi.org/10.1016/j.mgene.2016.11.005 2214-5400/© 2016 Elsevier B.V. All rights reserved.

RF might give a worse prognosis (van Venrooij et al., 2004; Song and Kang, 2010). Also, anti-cyclic citrullinated peptide (anti-CCP) antibodies are highly specific markers for RA diagnosis (van Gaalen et al., 2004), preceding the manifestation of disease symptoms, consequently provide a valuable diagnostic test in the early disease course (Rantapaa-Dahlqvist et al., 2003; Song and Kang, 2010) and are reported to become good predictors for the RA development (van Gaalen et al., 2004). This information suggests that these antibodies have a causative role in the RA pathogenesis (Balandraud et al., 2013; Kuhn et al., 2006). Though in many RA cases a course progressive joint disease is variable and character, and there is a need of consistent tests to assess RA outcome; in which around onethird of established RA cases are seronegative for diagnostically practical disease markers rheumatoid factor (RF) and antibodies against cyclic citrullinated peptide (ACCP). Furthermore, the sensitivities of both markers for RA are reported to be still lower in the diagnostically significant in early disease phase. These results indicate the need for further disease markers, especially for early RA and for ACCP-negative, RF-negative (seronegative) RA patients (Hussein et al., 2014; Somers et al., 2011).

S.Y. Elkhawaga et al. / Meta Gene 11 (2017) 58–63

Many inflammatory mediators involved in the pathology of RA, such as interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-α) and IL-6, are significantly over expressed in RA and are regulated by nuclear factor kappa B (NF-κB). NF-κB also regulates the differentiation of CD4+ T cells, particularly the T helper (Th) 17 cells, which have essential role in the pathogenesis of inflammation and autoimmunity (Dong, 2008; Okamoto and Kobayashi, 2011; Sun et al., 2013; Mellado et al., 2015). The tumor necrosis factor alpha- induced protein 3 (TNFAIP3), encodes ubiquitin-editing protein A20 which restricts the NF-κB activation in response to multiple signaling pathways, including those of IL-1, TNF α, NLR [Nod (nucleotide-binding oligomerization domain)-like receptor] ligands and Toll-like receptor (TLR) via its zinc finger domains (ZnF1–ZnF7) in C-terminus that supports E3 ubiquitin ligase activity and deubiquitinating (DUB) activity by removing its lysine-63-linked polyubiquitin chain, that is essential for its activation mediated by its OUT (ovarian tumor) domain in N-terminus. Subsequently, A20 promotes lysine-48-linked polyubiquitination of receptor interacting protein-1 (RIP-1) targeting it for proteasomal degradation, thereby fully interrupting NF-κB signaling (Boone et al., 2004; Vereecke et al., 2011; Hymowitz and Wertz, 2010; Wertz et al., 2004; Shembade et al., 2008). Various TNFAIP3 single nucleotide polymorphisms (SNPs) were found to be associated with the susceptibility to autoimmune disease (Lodolce et al., 2010; Coornaert et al., 2009; Musone et al., 2011). Genome-wide association studies (GWAS) have implicated TNFAIP3 locus are associated with susceptibility to many autoimmune and inflammatory diseases in different cohorts, including systemic lupus erythematosus (SLE), psoriasis, RA, diabetes type 1, and inflammatory bowel disorder (Elsby et al., 2010; Scherer et al., 2010). A20 protein participates in the negative regulation of inflammatory responses, and any alterations in the activity or expression of TNFAIP3 gene may influence the pathogenesis of RA (Tavares et al., 2010). Several studies have documented a strong association between TNFAIP3 polymorphisms and the risk of RA has been reported in different ethnic groups (Dieguez-Gonzalez et al., 2009; Hughes et al., 2010; Musone et al., 2011). TNFAIP3 gene maps to chromosome 6q23. The TNFAIP3 rs2230926 T NG, a nonsynonymous common coding single nucleotide polymorphism (SNP) in exon 3 of the gene, which introduces the amino acid substitution of phenylalanine to cysteine at amino acid position 127 in the ovarian tumor (OTU) domain of TNPAIP3 has been suggested to play a role in the inhibitory function of A20 enzyme (Coornaert et al., 2009). Furthermore, it has been reported that the Cys127 allele product (G) is slightly less effective at inhibiting NF-κB activation than the Phe127 allele product (T) and shows the strongest association with autoimmunity (Musone et al., 2008; Shimane et al., 2010). The allelic frequencies of genes often differ between populations. We aimed to assess the association between polymorphisms in the TNFAIP3 rs2230926 TNG and susceptibility, activity, and functional disability of RA in Egyptian patients. 2. Subjects and methods 2.1. Study subjects The present study included 82 RA patients (70 females and 12 males); all patients were diagnosed according to the 2010 EULAR/ACR criteria (Aletaha et al., 2010), age mean ± SD was 47.98 ± 10.67 years (range 23–72), their mean onset of disease was 39.32 ± 10.86 years and their mean disease duration was 8.66 ± 5.49 years. They were recruited from the outpatient and inpatient of Rheumatology, Rehabilitation, and physical medicine Departments; Faculty of Medicine Hospitals; Al-Azhar University, Egypt. Exclusion criteria included subjects with abnormal hepatic or renal functions, history of malignancy, pregnancy and lactation, alcohol abuse, and cases coupled with other autoimmune disorders. For comparison, 81 unrelated healthy controls were enrolled in the study (62 females and 19 males) from the same area, mean age was 46.21 ± 7.56 years (range 30–63), and any

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consanguinity was not present among the two groups. The study was approved by the ethical committee of the faculty of medicine hospitals, Al-Azhar University. Informed approval was obtained from all individuals included in the study. 2.2. Laboratory investigations Erythrocyte sedimentation rate in the first hour in mm/h was determined by Westergren method. Serum IgM RF and CRP were measured by latex turbidimetric immunoassay via BioSystems S.A. Costa Brava, 30.08030 Barcelona (Spain) kits. The IgM-RF and CRP were considered positive at values ≥ 30 IU/mL and 5 mg/L respectively. Anti-CCP (IgG) antibodies were measured by ELISA using the Immunoscan CCPLUS® test kit purchased from Euro-Diagnostica, Lundavägen 151, 212 24 Malmö (Sweden); and a concentration ≥25 U/mL was considered to be positive. According to the manufacturer instructions the assays were performed. 2.3. Clinical assessment Patients were subjected to complete history taking. As regards RA patients, disease activity was determined on the basis of The EULAR Disease Activity Score (DAS-28) involves a measure of the number of tender and swollen joints out of 28; the level of acute inflammatory markers (ESR or CRP) (Prevoo et al., 1995); these are entered into a specific formula and produce a score between 0 and 10. Functional disability was determined on the basis of parameters defined by the health assessment questionnaire (HAQ) (Maska et al., 2011). 2.4. DNA extraction and genotyping For all subjects, genomic DNA was extracted from EDTA – treated peripheral blood leukocytes with a DNA Blood Extraction kit GeneJET™ whole blood genomic DNA purification mini kit by Thermo Scientific, USA. DNA purity and concentration were measured at 260/280 absorbance by Thermo Scientific NanoDrop 2000 UV–Vis spectrophotometer, Wilmington, Delaware, USA. Samples with DNA concentration N10 ng/μL, 260/280 ratio between 1.7 and 1.9 were selected for genotyping. And then the DNA samples were frozen and stored at −80 °C until analysis. DNA of all subjects was genotyped for TNFAIP3 rs2230926 TN G polymorphism by Real Time-PCR using TaqMan® allele discrimination assay. The TNFAIP3 rs2230926 Probes and primers were designed by Applied Biosystems ID: rs2230926, C_7701116_10; Applied Biosystems, Foster City, Ca, USA. Amplification was performed in reaction volume 20 μL containing 10 μL of TaqMan® Genotyping Master Mix 2× , 0.5 μL of TaqMan® assay for SNP genotyping mix 40 ×, and 9.5 μL [DNA (20 ng in every well) + DNase free water]. Thermal cycling conditions were initiated by AmpliTaq Gold® polymerase activation at 95 °C for 10 min, followed by 45 cycles (denaturation at 95 °C for 15 s then annealing/extension at 60 °C for 1 min) and fluorescence was detected using ViiA7™ detector (Applied Biosystems, California, USA) and then analyzed with the manufacture's software. Nearly, 7% of the samples were amplified twice blindly for checking the accuracy of the results, which were found to be in harmony for all of the duplicate sets. All scatter plots were examined visually to confirm the accuracy of the genotyped records. 2.5. Statistical analysis All data were processed and analyzed using the Graph Pad Prism software version 6.1 (Graph Pad Software Inc., Ca, USA). The frequency of genotypes and alleles among cases was compared to those of controls using Fisher's exact test and the odd ratio (OR) with 95% confidence intervals (CI) was calculated using 2 × 2 contingency tables for estimating the association between certain genotypes and RA. Quantitative traits were compared using the unpaired t-test; the quantitative variables

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S.Y. Elkhawaga et al. / Meta Gene 11 (2017) 58–63

were articulated as average SD. Pearson's chi-square or Fisher's exact was used to comparing the distribution of several clinical items according to TNFAIP3 genotypes. Conformity with the HardyWeinberg law equilibrium (HWE) of genetic was assured by a no significant Chi-square test comparing the observed vs. the expected genotypes among cases and controls (P N 0.05). All of the tests twosided and the statistical significance were defined as (P b 0.05). 3. Results 3.1. The demographic and clinical characteristics of rheumatoid arthritis patients The demographic and clinical data of the RA group were shown in Table 1. There were no significant differences between RA patients and control subjects as regards age and sex distributions. 3.2. Distribution of TNFAIP3 rs2230926 TN G genotypes and alleles in RA patients and control groups The genotypes and alleles frequencies of TNFAIP3 rs2230926 TN G in RA patients and controls were illustrated in Table 2. The frequencies of TNFAIP3 rs2230926 T NG genotypes were in agreement with Hardy– Weinberg equilibrium (p N 0.05) in control and RA groups. Analyzes for a dominant copy of inheritance (TG + GG vs. TT) showed that RA cases had statistically significant higher frequency of the G allele carriers (TG + GG genotypes) compared to controls (35.37% vs. 14.82%, OR = 3.15, 95% CI = 1.47–6.74, P = 0.0036). Cases had statistically significant higher frequency of the TG genotype in RA cases compared to the controls (34.15% vs. 14.82%, OR = 3.04, 95% CI = 1.41–6.53, P = 0.0059). Regarding the allele frequencies, cases showed statistically significant higher frequency of the TNFAIP3 rs2230926 G allele compared to controls (18.30% vs. 7.40%, OR = 2.8, 95% CI = 1.38–5.69, P = 0.0045). 3.3. Relation between TNFAIP3 rs2230926 TNG polymorphism and clinical, and laboratory data of RA patients In Table 3, comparing RA patients subgroups related to their family history, gender, consanguinity, age of onset, disease duration, rheumatoid nodule, rheumatoid deformity, CRP, RF levels or status and anti-CCP levels Table 1 The demographic and clinical characteristics of rheumatoid arthritis patients compared to controls. Variable

Cases n = 82

Controls n = 81

Age (years) (M ± SD) Gender male/female n (%)/n (%)

47.98 ± 10.67 12 (14.63)/70 (85.37) 39.32 ± 10.86 5 (6.1) 11 (13.4) 8.66 ± 5.49 20 (24.39) 29 (35.37) 54.05 ± 30.37 19.27 ± 25.07 76.02 ± 2.57 427.4 ± 742.2 48 (58.54) 54 (65.85) 14.90 ± 7.023 6.23 ± 4.38 5.54 ± 2.8 5.08 ± 1.12 1.7 ± 0.68

46.21 ± 7.56 19 (23.46)/62 (76.54)

Age of onset (years) (M ± SD) Positive consanguinity n (%) Positive family history n (%) Disease duration (years) (M ± SD) Positive rheumatoid nodule n (%) Positive rheumatoid deformity n (%) ESR (mm/h) (M ± SD) CRP (mg/L) (M ± SD) RF (IU/mL) (M ± SD) Anti-CCP (U/mL) (M ± SD) Anti-CCP (+) ≥ 25 n (%) RF (+) ≥ 30 n (%) Number of tender joints (M ± SD) Number of swollen joints (M ± SD) VAS (M ± SD) DAS28-CRP (M ± SD) MHAQ score (M ± SD)

– – – – – – 14.00 ± 4.88 2.56 ± 1.73 12.99 ± 8.56 19.05 ± 3.16 0 (0) 0 (0) – – – – –

ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; RF: rheumatoid factor; ACPA: anti-cyclic citrullinated peptide (anti-CCP); VAS: visual analogue scale; DAS28: disease activity score 28 and MHAQ: modified health assessment questionnaire; (+): positive. Values are presented as mean ± SD, n (%).

Table 2 The genotypes and alleles frequency distributions of TNFAIP3 rs2230926 TNG in the RApatients and control groups. rs2230926 Genotypes TT TG GG TG + GG Alleles T G HWE

RA cases n (%)

Controls n (%)

OR (95% CI)

P value

53 (64.63) 28 (34.15) 1 (1.22) 29 (35.37)

69 (85.18) 12 (14.82) 0 (0.00) 12 (14.82)

1.00 – 3.04 (1.41–6.53) 0.0059⁎⁎

134 (81.71) 30 (18.29) χ2 = 1.66, P N 0.05

150 (92.59) 1.00 12 (7.41) 2.8 (1.38–5.69) χ2 = 0.52, P N 0.05

3.15 (1.47–6.74) 0.0036⁎⁎ – 0.0045⁎⁎

RA: rheumatoid arthritis; OR: odds ratio; CI: confidence intervals; HWE: Hardy–Weinberg equilibrium. ⁎⁎ Highly significant at P value b0.05.

or status, number of swollen joints (SJC), number of tender joints (TJC), visual analogue scale (VAS), RA disease activity score (DAS28), health assessment questionnaire (HAQ) score regarding their TNFAIP3 rs2230926 TNG genotypes showed that not any of these clinical and laboratory parameters had an association with the TNFAIP3 rs2230926 genotypes except for the rheumatoid deformity (P = 0.03) and health assessment questionnaire (HAQ) score (P = 0.004) that was statistically significant higher among cases carrying the TNFAIP3 rs2230926 G allele (TG + GG genotypes) compared with TNFAIP3 rs2230926 TT genotype. We stratified patients according to the presence of anti-CCP antibodies and RF and examined the association between TNFAIP3 rs2230926 T NG polymorphism and susceptibility for RA; we found no association between anti-CCP and RF levels or status in RA and genotype frequencies.

Table 3 Relation between TNFAIP3 rs2230926 TNG polymorphism and clinical, and laboratory data of RA patients. Variable

TT (53)

TG + GG (29)

P value

Age of onset (years) (M ± SD) Gender male/female n (%)/n (%)

40 ± 10.94 9 (16.98)/44 (83.02) 5 (9.43)/48 (90.57) 5 (9.43)/48 (90.57) 8.32 ± 5.85

38.0 ± 10.79 3 (10.35)/26 (89.65) 0 (0)/29 (100)

0.43 0.53

6 (20.69)/23 (79.31) 9.28 ± 4.79

0.18

14 (26.42)/39 (73.58) 14 (26.42)/39 (73.58) 52.8 ± 28.23 17.17 ± 20.51 75.73 ± 66.94 313.6 ± 552.2 31 (58.49)/22 (41.51) 33 (62.26)/20 (37.74) 14.38 ± 7.182 6.17 ± 4.82 5.91 ± 2.97 5.03 ± 1.14 1.57 ± 0.67

6 (20.69)/23 (79.31) 15 (51.72)/14 (48.28) 56.28 ± 32.77 23.09 ± 31.83 76.55 ± 54.81 635.5 ± 978.7 17 (58.62)/12 (41.38) 21 (72.41)/8 (27.59) 15.86 ± 6.74 6.35 ± 3.51 4.86 ± 2.36 5.17 ± 1.08 2.00 ± 0.61

Consanguinity +ve/−ve n (%)/n (%) Family history +ve/−ve n (%)/n (%) Disease duration (years) (M ± SD) Rheumatoid nodule +ve/−ve n (%)/n (%) Rheumatoid deformity +ve/−ve n (%)/n (%) ESR (mm/h) (M ± SD) CRP (mg/L) (M ± SD) RF (IU/mL) (M ± SD) Anti-CCP (U/mL) (M ± SD) Anti-CCP status +ve/−ve n (%)/n (%) RF status +ve/−ve n (%)/n (%) No. of tender joints (M ± SD) No. of swollen joints (M ± SD) VAS (M ± SD) DAS28-CRP (M ± SD) MHAQ score (M ± SD)

0.16

0.21 0.60 0.03⁎ 0.61 0.8 0.94 0.34 1 0.47 0.26 0.86 0.11 0.47 0.004⁎⁎

ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; RF: rheumatoid factor; ACPA: anti-cyclic citrullinated peptide (anti-CCP); VAS: visual analogue scale; DAS28: disease activity score 28 and MHAQ: modified health assessment questionnaire. Values are presented as mean ± SD, n (%). ⁎ Significant at P value b0.05. ⁎⁎ Highly significant at P value b0.05.

S.Y. Elkhawaga et al. / Meta Gene 11 (2017) 58–63 Table 4 Association of TNFAIP3 rs2230926 TNG polymorphism with joint deformity in RA patients.

rs2230926 Genotype

Allele

TT TG GG TG/GG T G

RA with joint deformity

RA without joint deformity

N

(%)

N

(%)

OR (95% CI)

P value

14 14 1 15 42 16

(48.28) (48.28) (3.44) (51.72) (72.41) (27.59)

39 14 0 14 92 14

(73.59) (26.41) 0 (26.41) (86.79) (13.21)

– 2.79 (1.07–7.28) – 2.99 (1.15–7.72) – 2.50 (1.12–5.60)

– 0.049⁎ – 0.030⁎ – 0.034⁎

RA: rheumatoid arthritis; OR: odds ratio; CI: confidence intervals. ⁎ Significant at P value b0.05.

3.4. Association of TNFAIP3 rs2230926 TN G polymorphism with joint deformity in RA patients In RA with joint deformity group, the frequencies of TG genotype were significantly increased compared to RA without joint deformity group (48.28% versus 26.41%). Subjects with TG genotype were significantly more likely to have joint deformity (OR = 2.79, 95% CI = 1.07–7.28, P = 0.049). The frequency of the G allele of TNFAIP3 rs2230926 was significantly increased in RA with joint deformity group compared to RA without joint deformity group (27.59% versus 13.21%). Carriers of the G allele (TG + GG genotype) were significantly more likely to develop joint deformity (OR = 2.99, 95% CI = 1.15–7.72, P = 0.030) as illustrated in Table 4.

3.5. Association between joint deformity and TNFAIP3 rs2230926 TN G polymorphism in seropositive and seronegative RA patients Carriers of the G allele (TG + GG genotype) were significantly more likely to develop joint deformity in seronegative RA patients for rheumatoid factor (RF) or anti cyclic citrullinated peptide (Anti-CCP) antibody than those carrying TT genotype (OR = 19, 95% CI = 1.65–218.6, P = 0.015, OR = 6.33, 95% CI = 1.20–33.4, P = 0.04 respectively). While in seropositive cases for RF or Anti-CCP antibody no significant difference showed between cases carrying the G allele and those carrying TT genotype (OR = 1.69, 95% CI = 0.56–5.11, P = 0.41, OR = 2, 95% CI = 0.61–6.82, P = 0.36 respectively) as illustrated in Table 5.

3.6. Association between joint deformity and RF (IU/mL), and disease duration (years) in RA patients In this study, significant association was found between higher serum RF levels and joint destruction. Also significant association was found between development of deformity and increased disease duration as in Table 6.

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4. Discussion In order to distinguish early RA patients with a mild disease course from those with an aggressive course, these require a serious search for diverse outcome markers, such as tests measuring cartilage and bone degradation. Therefore, our key guides to therapy at present time are the standard clinical and laboratory tests, like ESR, CRP and RF, and radiographs (Hussein et al., 2014; Aman et al., 2000). The novel finding in this study was the development of joint deformity was more likely to occur in seronegative RA patients for RF or anti-CCP who had TNFAIP3 rs2230926 (TG + GG genotype) than those with TNFAIP3 rs2230926 TT genotype. While in seropositive RA patients no significant difference between development of joint deformity among cases carrying the G allele (TG + GG genotypes) and those carrying TT genotype. In the present study, we have assessed the influence of abnormal laboratory investigation results to predict the progression of disease course. These results suggested that autoantibody-positive RA patients are both genetically and clinically distinct phenotypes, thus these autoantibodies have been participated in promoting the progress of structural damage in RA. This finding was explained by Jung et al. (2014) and Kocijan et al. (2013) who reported that the joint destruction in RA is initiated by autoimmunity and inflammation. In the present study, Egyptian RA patients showed significantly higher frequency of the TNFAIP3 rs2230926 G allele and TG + GG genotypes compared to the controls; with a relatively high odds ratio (OR = 3.15). Our results suggest that polymorphism in the TNFAIP3 rs2230926 gene was associated with susceptibility to RA. As a result, it seems likely that this region of A20 gene contains a number of genetic factors involved in RA. Some studies of TNFAIP3 in RA have demonstrated an association with multiple disease risk single nucleotide polymorphisms (SNPs), including rs6920220, rs2230926, and rs10499194 (Lee et al., 2012). Several studies have confirmed a strong association between rs2230926 and RA, and this SNP plays a functional role in the development of RA. Studies in the Caucasian cohort have been shown that genetic variants at the TNFAIP3 locus increase the susceptibility to RA (Orozco et al., 2009; Bowes et al., 2010). There is a close association between TNFAIP3 rs2230926 and increased risk for SLE and RA in the Japanese population (Shimane et al., 2010). In harmony study Lee et al. (2012), found an association between TNFAIP3 rs2230926 polymorphism and RA susceptibility in Europeans and Asians. However, these findings are not consistent with the study of (Zhu et al., 2015) in southern Chinese population who found just three cases with TNFAIP3 rs2230926 TG genotype in 50 RA samples; also found two cases with SNP rs2230926 TG genotype among 30 control individuals. Therefore, this preliminary results may designate a minor incidence of TNFAIP3 rs2230926 (TG/GG) genotype in Chinese RA patients. Also results were reported by Orozco et al. (2009) found that SNP rs2230926 is not independently associated with RA. In addition Kim et al. (2014) showed in the Korean population that rs2230926 in TNFAIP3 is not associated with RA susceptibility. Though, results from Zhang et al.

Table 5 Association between joint deformity and TNFAIP3 rs2230926 TNG polymorphism in seropositive and seronegative RA patients. Patients with joint deformity N = 29 Variable RF (IU/mL) Anti-CCP (U/mL)

≥30 b30 ≥25 b25

Patients without joint deformity N = 53

TG + GG

TT

TG + GG

TT

OR (95% CI)

P value

11 4 9 6

13 1 11 3

10 4 8 6

20 19 20 19

1.69 (0.56–5.11) 19 (1.65–218.6) 2 (0.61–6.82) 6.33 (1.20–33.4)

0.41 0.015⁎ 0.36 0.04⁎

RF: rheumatoid factor; anti-CCP: anti-cyclic citrullinated peptide; OR: odds ratio; CI: confidence intervals. ⁎ Significant at P value b0.05.

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Table 6 Association between joint deformity and RF (IU/mL), and disease duration (years) in RA patients. Joint deformity Variable

(+) N = 29

(−) N = 53

P value

RF (IU/mL), (M ± SD) Disease duration (years), (M ± SD) Positive RF (IU/mL), ≥30 (IU/mL)

94.19 ± 58.8 10.34 ± 6.23 24

66.07 ± 62.87 7.74 ± 4.86 30

0.046⁎ 0.039⁎ 0.027⁎

RF: rheumatoid factor; (+): present; (−): absent. ⁎ Significant at P value b0.05.

(2013) reported that TNFAIP3 rs2230926 is associated with risk of RA in the northern Chinese population, Hao et al. (2014) showed TNFAIP3 rs2230926 G allele was significantly increased in RA cases of southern Chinese Han population comparing with controls; furthermore, the comparison of genotypes frequency between patients and controls in TNFAIP3 rs2230926 was a significantly different. Zhang et al. (2014) showed that TNFAIP3 rs2230926 polymorphisms in the A20 gene region may be a susceptible factor for RA in the northern Chinese Han population. These contradictory results might be qualified to different ethnic origins, an environmental difference as well as problems related to research methodologies as the sufficiency of sample size and proper diagnostic methods, in addition to the differences in the potential contribution of patient populations (age and disease onset, female percentage, disease severity) might lead to dissimilar results. Regarding the activity and functional disability of RA; our study showed that various clinical feature had no association with the TNFAIP3 rs2230926 T NG polymorphism except for the HAQ that was significantly higher among cases with the G allele carriage (TG + GG genotypes) than those with the TT genotype. In fact this finding is remarkable; thus though the disease activity looked apparently with no difference among patients with different genetic background through the clinical assessment of the disease, it was actually significantly different through the assessment of patient quality of life (QoL). The rs6920220, rs10499194, and rs2230926 polymorphisms have also been reported to be associated with the severity of autoimmune diseases (Lee et al., 2012). Our data revealed that the rheumatoid deformity was significantly higher among cases carrying the TNFAIP3 rs2230926 G allele (TG + GG genotypes) compared with TNFAIP3 rs2230926 TT genotype. Thus, patients who are carrier for the G allele of rs2230926 in TNFAIP3 may develop a more severe form of the disease with increased disease disability. We might consider that Egyptian RA patients who are G allele carriers are expressing a loss of function protein, A20 which participates in negative regulation inflammatory responses (Tavares et al., 2010); consequently leads to excessive immune activity, and thus enhanced autoreactivity. Moreover, Chinese RA cases whose TNFAIP3 rs2230926 G allele carrier (TG + GG genotypes) had poor functional outcome (Zhu et al., 2015). Furthermore, A20-deficient mice have been reported to exhibit systematic inflammation, joints damage, and to develop autoimmunity (Lee et al., 2000). In our study, the results showed there was a significant association between positive RF and hand deformity. This is in agreement with de Vries-Bouwstra et al. (2008) and Terao et al. (2015) who described that there was a significant association between positive RF and hand deformities. This result is due to that those RF-positive patients with RA have more aggressive and erosive joint disease and extraarticular manifestations than those who are RF-negative. Our results revealed that the association between the TNFAIP3 rs2230926 TNG polymorphism and risk of RA is not dependent on the presence of serum autoantibodies (RF or anti-CCP). This is in agreement with studies for the Korean population (Kim et al., 2014) who found that genotype in the TNFAIP3 rs2230926 gene was not associated with the presence of anti-CCP or RF antibody and risk of RA; in the Chinese population Zhu et al. (2015) found that there was no correlation between the rs2230926 SNP and the presence of anti-CCP antibody. In

contrast to these reports, a previous study in Japanese subjects found that the minor G allele increased the risk of RA in patients with RF and anti-CCP antibody-positive patients compared with RF and anti-CCP antibody-negative patients (Shimane et al., 2010). This study has several limitations which require consideration. First, the findings were preliminary results from a limited number of cases and controls. Second, the study only analyzed the association of one SNP polymorphism RA disease. Third, the lack of replicative patients and controls compromises the evidentiary assessment of the results. Therefore, additional studies in large patients and controls of different ethnicity with longitudinal continue of the patients and more polymorphisms in TNFAIP3 gene are required to explore the possible effect of others A20 polymorphisms on RA disease. Moreover, a combination of genetic factors collectively with environmental exposures should be careful. 5. Conclusion The present study suggested that TNFAIP3 rs2230926 G allele is associated with susceptibility to RA, and functional disability in patients with rheumatoid arthritis. This may provide a good marker for the diagnosis of RA susceptibility and help in the prediction of disease severity. Conflict of interest None. Acknowledgments Authors are grateful for the staff members of Rheumatology Department, Faculty of medicine Hospitals, Al-Azhar University, Cairo, Egypt for their help in selecting cases under the study. References Aletaha, D., Neogi, T., Silman, A.J., Funovits, J., Felson, D.T., Bingham III, 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., Hobbs, K., Huizinga, T.W., Kavanaugh, A., Kay, J., Kvien, T.K., Laing, T., Mease, P., Menard, H.A., Moreland, L.W., Naden, R.L., Pincus, T., Smolen, J.S., Stanislawska-Biernat, E., Symmons, D., Tak, P.P., Upchurch, K.S., Vencovsky, J., Wolfe, F., Hawker, G., 2010. 2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Ann. Rheum. Dis. 69, 1580–1588. Aman, S., Paimela, L., Leirisalo-Repo, M., Risteli, J., Kautiainen, H., Helve, T., Hakala, M., 2000. Prediction of disease progression in early rheumatoid arthritis by ICTP, RF and CRP. A comparative 3-year follow-up study. Rheumatology (Oxford) 39, 1009–1013. Balandraud, N., Picard, C., Reviron, D., Landais, C., Toussirot, E., Lambert, N., Telle, E., Charpin, C., Wendling, D., Pardoux, E., Auger, I., Roudier, J., 2013. HLA-DRB1 genotypes and the risk of developing anti citrullinated protein antibody (ACPA) positive rheumatoid arthritis. PLoS One 8, e64108. Boone, D.L., Turer, E.E., Lee, E.G., Ahmad, R.C., Wheeler, M.T., Tsui, C., Hurley, P., Chien, M., Chai, S., Hitotsumatsu, O., McNally, E., Pickart, C., Ma, A., 2004. The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses. Nat. Immunol. 5, 1052–1060. Bowes, J., Lawrence, R., Eyre, S., Panoutsopoulou, K., Orozco, G., Elliott, K.S., Ke, X., Morris, A.P., Thomson, W., Worthington, J., Barton, A., Zeggini, E., 2010. Rare variation at the TNFAIP3 locus and susceptibility to rheumatoid arthritis. Hum. Genet. 128, 627–633. Coornaert, B., Carpentier, I., Beyaert, R., 2009. A20: central gatekeeper in inflammation and immunity. J. Biol. Chem. 284, 8217–8221. de Vries-Bouwstra, J.K., Goekoop-Ruiterman, Y.P., Verpoort, K.N., Schreuder, G.M., Ewals, J.A., Terwiel, J.P., Ronday, H.K., Kerstens, P.J., Toes, R.E., de Vries, R.R., Breedveld, F.C., Dijkmans, B.A., Huizinga, T.W., Allaart, C.F., 2008. Progression of joint damage in early rheumatoid arthritis: association with HLA-DRB1, rheumatoid factor, and anti-citrullinated protein antibodies in relation to different treatment strategies. Arthritis Rheum. 58, 1293–1298. Dieguez-Gonzalez, R., Calaza, M., Perez-Pampin, E., Balsa, A., Blanco, F.J., Canete, J.D., Caliz, R., Carreno, L., de la Serna, A.R., Fernandez-Gutierrez, B., Ortiz, A.M., HerreroBeaumont, G., Pablos, J.L., Narvaez, J., Navarro, F., Marenco, J.L., Gomez-Reino, J.J., Gonzalez, A., 2009. Analysis of TNFAIP3, a feedback inhibitor of nuclear factorkappaB and the neighbor intergenic 6q23 region in rheumatoid arthritis susceptibility. Arthritis Res. Ther. 11, R42. Dong, C., 2008. TH17 cells in development: an updated view of their molecular identity and genetic programming. Nat. Rev. Immunol. 8, 337–348.

S.Y. Elkhawaga et al. / Meta Gene 11 (2017) 58–63 Elsby, L.M., Orozco, G., Denton, J., Worthington, J., Ray, D.W., Donn, R.P., 2010. Functional evaluation of TNFAIP3 (A20) in rheumatoid arthritis. Clin. Exp. Rheumatol. 28, 708–714. Firestein, G.S., 2003. Evolving concepts of rheumatoid arthritis. Nature 423, 356–361. Gourraud, P.A., Boyer, J.F., Barnetche, T., Abbal, M., Cambon-Thomsen, A., Cantagrel, A., Constantin, A., 2006. A new classification of HLA-DRB1 alleles differentiates predisposing and protective alleles for rheumatoid arthritis structural severity. Arthritis Rheum. 54, 593–599. Hao, G., Li, Y., Liu, J., Wo, M., 2014. TNFAIP3 Rs2230926 polymorphisms in rheumatoid arthritis of southern Chinese Han population: a case-control study. Int. J. Clin. Exp. Pathol. 7, 8958–8961. Hughes, L.B., Reynolds, R.J., Brown, E.E., Kelley, J.M., Thomson, B., Conn, D.L., Jonas, B.L., Westfall, A.O., Padilla, M.A., Callahan, L.F., Smith, E.A., Brasington, R.D., Edberg, J.C., Kimberly, R.P., Moreland, L.W., Plenge, R.M., Bridges Jr., S.L., 2010. Most common single-nucleotide polymorphisms associated with rheumatoid arthritis in persons of European ancestry confer risk of rheumatoid arthritis in African Americans. Arthritis Rheum. 62, 3547–3553. Hussein, Y.M., Mohamed, R.H., El-Shahawy, E.E., Alzahrani, S.S., 2014. Interaction between TGF-beta1 (869C/T) polymorphism and biochemical risk factor for prediction of disease progression in rheumatoid arthritis. Gene 536, 393–397. Hymowitz, S.G., Wertz, I.E., 2010. A20: from ubiquitin editing to tumour suppression. Nat. Rev. Cancer 10, 332–341. Jung, S.M., Kim, K.W., Yang, C.W., Park, S.H., Ju, J.H., 2014. Cytokine-mediated bone destruction in rheumatoid arthritis. J. Immunol. Res. 2014, 263625. Kim, S.K., Choe, J.Y., Bae, J., Chae, S.C., Park, D.J., Kwak, S.G., Lee, S.S., 2014. TNFAIP3 gene polymorphisms associated with differential susceptibility to rheumatoid arthritis and systemic lupus erythematosus in the Korean population. Rheumatology (Oxford) 53, 1009–1013. Kocijan, R., Harre, U., Schett, G., 2013. ACPA and bone loss in rheumatoid arthritis. Curr. Rheumatol. Rep. 15, 366. Kuhn, K.A., Kulik, L., Tomooka, B., Braschler, K.J., Arend, W.P., Robinson, W.H., Holers, V.M., 2006. Antibodies against citrullinated proteins enhance tissue injury in experimental autoimmune arthritis. J. Clin. Invest. 116, 961–973. Lee, E.G., Boone, D.L., Chai, S., Libby, S.L., Chien, M., Lodolce, J.P., Ma, A., 2000. Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice. Science 289, 2350–2354. Lee, Y.H., Bae, S.C., Choi, S.J., Ji, J.D., Song, G.G., 2012. Associations between TNFAIP3 gene polymorphisms and rheumatoid arthritis: a meta-analysis. Inflamm. Res. 61, 635–641. Lodolce, J.P., Kolodziej, L.E., Rhee, L., Kariuki, S.N., Franek, B.S., McGreal, N.M., Logsdon, M.F., Bartulis, S.J., Perera, M.A., Ellis, N.A., Adams, E.J., Hanauer, S.B., Jolly, M., Niewold, T.B., Boone, D.L., 2010. African-derived genetic polymorphisms in TNFAIP3 mediate risk for autoimmunity. J. Immunol. 184, 7001–7009. MacGregor, A.J., Snieder, H., Rigby, A.S., Koskenvuo, M., Kaprio, J., Aho, K., Silman, A.J., 2000. Characterizing the quantitative genetic contribution to rheumatoid arthritis using data from twins. Arthritis Rheum. 43, 30–37. Maska, L., Anderson, J., Michaud, K., 2011. Measures of functional status and quality of life in rheumatoid arthritis: Health Assessment Questionnaire Disability Index (HAQ), Modified Health Assessment Questionnaire (MHAQ), Multidimensional Health Assessment Questionnaire (MDHAQ), Health Assessment Questionnaire II (HAQ-II), Improved Health Assessment Questionnaire (Improved HAQ), and Rheumatoid Arthritis Quality of Life (RAQoL). Arthritis Care. Res. 63 (Suppl. 11), S4–13. Mellado, M., Martinez-Munoz, L., Cascio, G., Lucas, P., Pablos, J.L., Rodriguez-Frade, J.M., 2015. T cell migration in rheumatoid arthritis. Front. Immunol. 6, 384. Musone, S.L., Taylor, K.E., Lu, T.T., Nititham, J., Ferreira, R.C., Ortmann, W., Shifrin, N., Petri, M.A., Kamboh, M.I., Manzi, S., Seldin, M.F., Gregersen, P.K., Behrens, T.W., Ma, A., Kwok, P.Y., Criswell, L.A., 2008. Multiple polymorphisms in the TNFAIP3 region are independently associated with systemic lupus erythematosus. Nat. Genet. 40, 1062–1064. Musone, S.L., Taylor, K.E., Nititham, J., Chu, C., Poon, A., Liao, W., Lam, E.T., Ma, A., Kwok, P.Y., Criswell, L.A., 2011. Sequencing of TNFAIP3 and association of variants with multiple autoimmune diseases. Genes Immun. 12, 176–182. Okamoto, H., Kobayashi, A., 2011. Tyrosine kinases in rheumatoid arthritis. J. Inflamm. (Lond.) 8, 21. Orozco, G., Hinks, A., Eyre, S., Ke, X., Gibbons, L.J., Bowes, J., Flynn, E., Martin, P., Wilson, A.G., Bax, D.E., Morgan, A.W., Emery, P., Steer, S., Hocking, L., Reid, D.M., Wordsworth, P., Harrison, P., Thomson, W., Barton, A., Worthington, J., 2009.

63

Combined effects of three independent SNPs greatly increase the risk estimate for RA at 6q23. Hum. Mol. Genet. 18, 2693–2699. Prevoo, M.L., van ‘ t Hof, M.A., Kuper, H.H., van Leeuwen, M.A., van de 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. Rantapaa-Dahlqvist, S., de Jong, B.A., Berglin, E., Hallmans, G., Wadell, G., Stenlund, H., Sundin, U., van Venrooij, W.J., 2003. Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum. 48, 2741–2749. Salama, A., Elshazli, R., Elsaid, A., Settin, A., 2014. Protein tyrosine phosphatase non-receptor type 22 (PTPN22) +1858CNT gene polymorphism in Egyptian cases with rheumatoid arthritis. Cell. Immunol. 290, 62–65. Scherer, H.U., van der Linden, M.P., Kurreeman, F.A., Stoeken-Rijsbergen, G., Cessie, S., Huizinga, T.W., van der Helm-van Mil, A.H., Toes, R.E., 2010. Association of the 6q23 region with the rate of joint destruction in rheumatoid arthritis. Ann. Rheum. Dis. 69, 567–570. Shembade, N., Harhaj, N.S., Parvatiyar, K., Copeland, N.G., Jenkins, N.A., Matesic, L.E., Harhaj, E.W., 2008. The E3 ligase Itch negatively regulates inflammatory signaling pathways by controlling the function of the ubiquitin-editing enzyme A20. Nat. Immunol. 9, 254–262. Shimane, K., Kochi, Y., Horita, T., Ikari, K., Amano, H., Hirakata, M., Okamoto, A., Yamada, R., Myouzen, K., Suzuki, A., Kubo, M., Atsumi, T., Koike, T., Takasaki, Y., Momohara, S., Yamanaka, H., Nakamura, Y., Yamamoto, K., 2010. The association of a nonsynonymous single-nucleotide polymorphism in TNFAIP3 with systemic lupus erythematosus and rheumatoid arthritis in the Japanese population. Arthritis Rheum. 62, 574–579. Somers, K., Geusens, P., Elewaut, D., De, K.F., Rummens, J.L., Coenen, M., Blom, M., Stinissen, P., Somers, V., 2011. Novel autoantibody markers for early and seronegative rheumatoid arthritis. J. Autoimmun. 36, 33–46. Song, Y.W., Kang, E.H., 2010. Autoantibodies in rheumatoid arthritis: rheumatoid factors and anticitrullinated protein antibodies. QJM 103, 139–146. Sun, S.C., Chang, J.H., Jin, J., 2013. Regulation of nuclear factor-kappaB in autoimmunity. Trends Immunol. 34, 282–289. Tavares, R.M., Turer, E.E., Liu, C.L., Advincula, R., Scapini, P., Rhee, L., Barrera, J., Lowell, C.A., Utz, P.J., Malynn, B.A., Ma, A., 2010. The ubiquitin modifying enzyme A20 restricts B cell survival and prevents autoimmunity. Immunity 33, 181–191. Terao, C., Yamakawa, N., Yano, K., Markusse, I.M., Ikari, K., Yoshida, S., Furu, M., Hashimoto, M., Ito, H., Fujii, T., Ohmura, K., Murakami, K., Takahashi, M., Hamaguchi, M., Tabara, Y., Taniguchi, A., Momohara, S., Raychaudhuri, S., Allaart, C.F., Yamanaka, H., Mimori, T., Matsuda, F., 2015. Rheumatoid factor is associated with the distribution of hand joint destruction in rheumatoid arthritis. Arthritis Rheumatol. 67, 3113–3123. van Gaalen, F.A., Linn-Rasker, S.P., van Venrooij, W.J., de Jong, B.A., Breedveld, F.C., Verweij, C.L., Toes, R.E., Huizinga, T.W., 2004. Autoantibodies to cyclic citrullinated peptides predict progression to rheumatoid arthritis in patients with undifferentiated arthritis: a prospective cohort study. Arthritis Rheum. 50, 709–715. van Venrooij, W.J., Vossenaar, E.R., Zendman, A.J., 2004. Anti-CCP antibodies: the new rheumatoid factor in the serology of rheumatoid arthritis. Autoimmun. Rev. 3 (Suppl. 1), S17–S19. Vereecke, L., Beyaert, R., van L, G., 2011. Genetic relationships between A20/TNFAIP3, chronic inflammation and autoimmune disease. Biochem. Soc. Trans. 39, 1086–1091. Wertz, I.E., O'Rourke, K.M., Zhou, H., Eby, M., Aravind, L., Seshagiri, S., Wu, P., Wiesmann, C., Baker, R., Boone, D.L., Ma, A., Koonin, E.V., Dixit, V.M., 2004. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling. Nature 430, 694–699. Zhang, X., Li, W., Zhang, X., Zhang, X., Jiang, L., Guo, Y., Zhang, J., Wang, X.F., Liang, Z.F., 2013. The association of single nucleotide polymorphisms in TNFAIP3 with rheumatoid arthritis in the Chinese population. J Immunol]–>Eur. J. Immunol. 38, 214–220. Zhang, X., Li, W., Zhang, X., Zhao, L., Zhang, X., Jiang, L., Guo, Y., Zhang, J., Liang, Z., Wang, X., 2014. Single nucleotide polymorphisms in TNFAIP3 were associated with the risks of rheumatoid arthritis in northern Chinese Han population. BMC Med. Genet. 15, 56. Zhu, L., Wang, L., Wang, X., Zhou, L., Liao, Z., Xu, L., Wu, H., Ren, J., Li, Z., Yang, L., Chen, S., Li, B., Wu, X., Zhou, Y., Li, Y., 2015. Characteristics of A20 gene polymorphisms and clinical significance in patients with rheumatoid arthritis. J. Transl. Med. 13, 215.