Signal transducer and activator of transcription 4 (STAT4) G>T gene polymorphism in Egyptian cases with rheumatoid arthritis

Human Immunology 75 (2014) 863–866

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Signal transducer and activator of transcription 4 (STAT4) G>T gene polymorphism in Egyptian cases with rheumatoid arthritis Ahmad Settin a, Afrah Salama b, Rami Elshazli b,⇑ a b

Genetics Unit, Children Hospital, Mansoura University, Mansoura, Egypt Department of Biochemistry, Faculty of Science, Tanta University, Tanta, Egypt

a r t i c l e

i n f o

Article history: Received 30 March 2014 Accepted 19 June 2014 Available online 28 June 2014 Keywords: Rheumatoid arthritis Gene polymorphism STAT4 Anti-CCP

a b s t r a c t Background: The gene encoding signal transducer and activator of transcription 4 (STAT4) has been reported to be associated with rheumatoid arthritis (RA) in several populations. This work aimed at assessing the association of STAT4 G>T gene polymorphism with the susceptibility, activity and functional disability of RA in Egyptian subjects. Subjects and methods: This study included 112 unrelated RA Egyptian patients who were compared to 122 healthy unrelated individuals taken from the same locality. For all subjects, DNA was genotyped for STAT4 G>T (rs7574865) polymorphism using the PCR-RFLP technique. Antibodies to cyclic citrullinated peptides (anti-CCP) were measured by enzyme-linked immunosorbent assay (ELISA). Results: Cases showed a significantly higher frequency of the STAT4 T allele carriage (GT+TT genotypes) compared to controls (51.8% vs. 31.1%, OR = 2.37, 95% CI = 1.39–4.05, p = 0.001). Also the frequency of the STAT4 T allele was significantly higher among cases compared to controls (30.4% vs. 16.8%, OR = 2.16, 95% CI = 1.39–3.35, p = 0.001). Cases positive to the STAT4 T allele (GT+TT genotypes) showed no significant difference compared to those with the GG genotype regarding their clinical and immune parameters. Nonetheless, they showed a more functional disability presented in their significantly higher health assessment questionnaire (HAQ) score (p = 0.02). Conclusions: This study gives an extra evidence to the association of the STAT4 T allele with the susceptibility and functional disability of RA. Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

1. Introduction Rheumatoid arthritis (RA) is the most common chronic autoimmune disease, affecting 1% of the adult population worldwide. It is characterized by chronic inflammation and destruction of the synovial joints, leading to progressive joint damage, and it is associated with significant disability and early mortality [1]. The etiology of RA is complex, and it is not completely understood. However, it is known that RA risk is probably influenced by an interaction between environmental and genetic factors [2,3]. Human leukocyte antigen (HLA) class II molecules were portrayed as being the most powerful genetic factors of RA identified to date. However, family studies suggest that this association accounts for only one-third of genetic susceptibility, and that non-HLA genes are also involved [4].

⇑ Corresponding author. E-mail address: [email protected] (R. Elshazli).

The polymorphism pertinent to the signal transducer and activator of transcription 4 (STAT4) gene seems to be one of the best examples of a non-MHC common susceptibility allele for autoimmunity [5]. STAT4 is expressed in activated peripheral blood monocytes, dendritic cells and macrophages at the sites of inflammation [6]. STAT4 transmits signals induced by interleukin-12 (IL12), interleukin-23 (IL-23) and interferon-c (IFN-c), which are key cytokines and play important roles in the development of autoimmune diseases [7,8]. The STAT4 gene maps to chromosome 2q33 and encodes a transcription factor, which plays pivotal roles in the differentiation and proliferation of both T helper 1 (Th1) and T helper 17 (Th17) cells [8]. Since Th1 and Th17 lineages are crucial effectors in chronic inflammatory disorders, STAT4 gene may play an important role in the pathogenesis of autoimmune diseases. To date, the SNP rs7574865 in STAT4 gene has been reported to be associated with an increased risk for diverse complex autoimmune diseases in different ethnic populations, such as RA [9–13]. Knowing that the allelic frequencies of genes often differ substantially between populations; we were interested to test the association

http://dx.doi.org/10.1016/j.humimm.2014.06.013 0198-8859/Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

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A. Settin et al. / Human Immunology 75 (2014) 863–866

of this seemingly important STAT4 G>T polymorphism with RA in Egyptian cases.

informed consent was obtained from all participants before their enrollment into the study.

2. Subjects and methods

2.2. Statistical analysis

This study included 112 RA patients (93 females and 19 males). All fulfilled the criteria for RA set by the American College of Rheumatology classification [14]. Their age mean ± SD was 47.6 ± 10.1 years. They were recruited from the Outpatient Clinic of Rheumatology and Rehabilitation department; Mansoura University Hospitals, Egypt. Exclusion criteria included cases with abnormal renal or hepatic functions, history of malignancy, alcohol abuse, pregnancy and lactation and cases associated with other autoimmune disorders. For comparison, another control sample was taken in the form of 122 healthy unrelated blood donors (99 females and 23 males) from the same locality. Their age mean ± SD was 42.3 ± 10.3 years. Disease activity was determined on the basis of multiply defined parameters including the swollen joint count (SJC), tender joint count (TJC), Disease Activity Score 28 (DAS28), C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) [15,16]. Functional disability was determined on the basis of health assessment questionnaire (HAQ) defined parameters that comprises eight general categories including: dressing and grooming, arising, eating, walking, hygiene, reach, grip and other activities. Each of which consists of one or more specific questions. Each question is scored either (0): ‘‘without any difficulty’’, (1): ‘‘with some difficulty’’, (2): ‘‘with much difficulty’’ and (3): ‘‘unable to do’’ with a summed score ranging 0–24 [17,18]. C-reactive protein (CRP) and rheumatoid factor (RF) were estimated by semi-quantitative latex agglutination with AVITEX-CRP and AVITEX-RF kits (Omega Diagnostics, Alva, Scotland, UK). Serum was considered positive when the titer is 6.0 mg/L for CRP and 8.0 IU/ml for RF. Anti-CCP was detected in serum samples using enzyme-linked immunosorbent assay (ELIZA) kit (Orgentec Diagnostics, Mainz, Germany). A concentration P20 U/ml was considered positive. For all participants, genotyping of the STAT4 G>T gene rs7574865 SNP was performed via the polymerase chain reaction-restriction fragment length polymorphism method (PCRRFLP), using the following forward (50 -AAA GAA GTG GGA TAA AAA GAA GTT TG-30 ) and reverse (50 -CCA CTG AAA TAA GAT AAC CAC TGT-30 ) primers [12]. PCR mixture contained 100 ng DNA, 10 pmol of each primer, 50 lm of dNTPs, 1X PCR buffer with MgCl2, 1 U Taq DNA polymerase and H2O up to 25 ll. PCR amplification protocol consisted of initial denaturation at 95 °C for 5 min, 30 cycles of amplification which included denaturation at 94 °C for 30 s, annealing at 52 °C for 60 s and extension at 72 °C for 60 s, and final extension at 72 °C for 7 min. The amplified product was digested with FastDigest HpaI restriction enzyme (Fermentas, Germany). The digestion product was applied on a 2% agarose gel stained with ethidium bromide permitting the differentiation of the following restriction pattern. The amplification of the STAT4 generate a 147 bp fragment. The G allele was not digested; so the GG genotype appeared as a single band develops at 147 bp while the T allele was cut into 122 and 25 bp fragments; so the TT genotype appeared as single band develops at 122 bp (25 bp fragment was too small to be detected); and the GT genotype appeared as two bands; one developed at 147 bp, and the other at 122 bp.

Data were processed and analyzed using the Statistical Package of Social Science (SPSS, version 17.0). The frequencies of studied genotypic and allelic variants among cases were compared to that of controls using Fisher’s exact test and odds ratio (OR) with the 95% confidence interval (95% CI). Quantitative traits were compared using the Student t-test while nominal traits were compared using the Chi square test. Conformity with the Hardy Weinberg law of genetic equilibrium (HWE) was assured by a non-significant Chi square test comparing the observed versus the expected genotype frequencies in cases and controls. A minimum level of statistical significance was considered at a p level of <0.05.

2.1. Ethical approval and informed consent The study was commenced on after obtaining an approval from the university ethical and scientific committees. In addition, an

3. Results The demographic and clinical data of the RA patients showed that 38 cases (33.9%) had a positive family history of rheumatoid arthritis and 50 cases (44.6%) had a positive consanguinity. Analysis of laboratory markers revealed that 104 cases (92.8%) were positive for anti-cyclic citrullinated peptide (anti-CCP) while 83 cases (74.1%) were positive for rheumatoid factor (RF). The genotypic and allelic frequencies of the STAT4 G>T gene SNP in RA patients and controls are shown in Table 1. The frequencies of STAT4 genotypes were in agreement with Hardy–Weinberg equilibrium in both RA and control groups (p > 0.05). Testing for the dominant model of inheritance (GT+TT vs. GG) showed that cases had a significantly higher frequency of the T allele carriers (GT+TT genotypes) compared to controls (51.8% vs. 31.1%, OR = 2.37, 95% CI = 1.39–4.05, p = 0.001). Cases had a significantly higher frequency of the GT genotype in RA cases compared to the controls (42.9% vs. 28.7%, OR = 2.13, 95% CI = 1.23–3.71, p = 0.008). Testing for the recessive model (TT vs. GG+GT), cases showed a significantly higher frequency of the TT homozygous genotype compared to the controls (8.9% vs. 2.4%, OR = 3.89, 95% CI = 1.04–14.52, p = 0.04). Regarding the allelic frequencies, cases showed a significantly higher frequency of the STAT4 T allele compared to controls (30.4% vs. 16.8%, OR = 2.16; 95% CI = 1.39–3.35, p = 0.001) (Table 1).

Table 1 The genotype and allele frequencies of STAT4 G>T in the RA patients and control groups. RA patients n (%)

Controls n (%)

Genotypes GG GT TT

54 (48.2) 48 (42.9) 10 (8.9)

84 (68.9) 35 (28.7) 3 (2.4)

Alleles G T

156 (69.6) 68 (30.4)

203 (83.2) 41 (16.8)

HWE

v2 = 0.02, p > 0.05

v2 = 0.08, p > 0.05

Statistics Recessive Dominant Codominant Allele

TT vs. GG+GT GT+TT vs. GG GT vs. GG TT vs. GG T vs. G

OR (95% CI)

p

3.89 2.37 2.13 5.19 2.16

0.04* 0.001** 0.008* 0.02* 0.001**

(1.04–14.52) (1.39–4.05) (1.23–3.71) (1.36–19.69) (1.39–3.35)

OR: odds ratio; CI: confidence intervals; HWE: Hardy–Weinberg equilibrium. * p < 0.05 = significant. ** p < 0.001 = highly significant.

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Comparing cases subgroups related to their age of onset, disease duration, gender, consanguinity, family history, rheumatoid deformity, rheumatoid nodule, number of swollen joints (SJC), number of tender joints (TJC), visual analogue scale (VAS), RA disease activity score (DAS28), health assessment questionnaire (HAQ) score, Creactive protein (CRP) status, rheumatoid factor (RF) status and anti-cyclic citrullinated peptide (anti-CCP) status regarding their STAT4 G>T genotypes (Table 2), showed that none of these clinical and laboratory parameters had an association with the STAT4 genotypes except for the HAQ score that was significantly higher among cases carrying the STAT4 T allele (GT+TT genotypes) compared with STAT4 GG genotype (p = 0.02).

4. Discussion This study is probing into the genetic and immune basis of RA in Egyptian patients noting that about one third of these cases had a positive family history of the disease and positive parental consanguinity. In recent years, numerous studies have tested the association between various candidate genes and the development of RA in patients of different ethnic origins [12,19,20]. Egyptian cases with RA showed significantly higher frequency of the STAT4 T allele carriage coping with the dominant model of inheritance; with a relatively high odds ratio (OR = 2.37). Recent meta-analysis showed that STAT4 rs7574865 T allele carriage was significantly associated with RA in Asian, Caucasian, Latin American and African subjects with an OR = 1.34, 1.36, 1.46 and 1.79 respectively which was slightly lower than the OR found in our study for the same genotypes [10]. In addition, a recent genome wide association study (GWAS) has also documented that this polymorphism is significantly associated with RA [19]. In another study done among Egyptian RA patients recruited from the eastern province of Egypt, Mohamed et al. reported a significant increase of STAT4 GT – but not the TT genotype – with an overall significant increase of the STAT4 T allele in RA cases compared to controls [11]. Similar studies demonstrating an association of RA with the STAT4 T allele in a dominant inheritance model included that done among Dutch [21], Spanish [22], Japanese [23], Chinese [20], Greek [12], Tunisian [24] and Colombian [25] subjects. STAT4 was shown to be abundantly expressed in synovial macrophages in patients with RA [6]. It was speculated to mediate the IL-12 signaling that is critical for development of protective immunity in intracellular infection which is dependent on the induction of Th1 responses and INF-c

production [7] inducing a protective role against joint damage progression [8]. In spite of the above evidence, we have encountered a single study stating a non-association of STAT4 T allele and RA among African-American subjects [26]. They explained this finding might be due to the confounding factor of the population admixture of cases with African-American ethnicity. They also had commented that – given the limited region of the gene they examined – it was possible that another region of STAT4 might be associated with RA susceptibility in populations of African ancestry. In this respect, we would – in addition – speculate that these contradictory results might be attributable to other environmental interactive factors. However, we would also recommend an additional study into the entire gene sequence in different cases from diverse ethnicities taking into consideration other interactive genetic polymorphisms. Regarding the activity and disability of RA, our study showed that all parameters had no association with the STAT4 rs7574865 polymorphism except for the HAQ score that was significantly higher among cases with the T allele carriage (GT + TT genotypes) than other cases. In fact the finding of a significant association of STAT4 T allele carriage with the HAQ score is interesting being illustrative of the genetic effect on the disease presentation through the assessment of patient quality of life (QoL) rather than from the clinical assessment of the disease. This was confirmed by the finding of Shen et al. who had reported an association of the STAT4 rs7574865 GT genotype with the disease activity among Chinese patients [20]. Our results revealed that the association between the STAT4 rs7574865 polymorphism and RA is not dependent of the presence of positive autoantibodies (RF and anti-CCP). This finding is conforming with that previously found in another study done among Dutch subjects showing no evidence of association between the minor T allele of STAT4 rs7574865 SNP and antibody positivity [21]. However, it is worth noting that most of our RA cases were seropositive for the anti-CCP that made the assessment of its association with risk genotypes difficult and non-conclusive. Therefore, authors do document some limitations of the study in the form of its relatively small sample size in addition to the lack of data concerning the STAT4 gene expression in studied cases. In this respect, we recommend undertaking a wider scale study of a large sample size including different disease phenotypes; investigating their genetic STAT4 polymorphisms along with its expression state in the blood and at the site of inflammation.

Table 2 Genotype frequencies of STAT4 G>T polymorphism stratified by the clinical items of rheumatoid arthritis.

Age of onset (years) Gender male/female Consanguinity +ve/ve Family history +ve/ve Disease duration (years) Number of swollen joints Number of tender joints Rheumatoid deformity +ve/ve Rheumatoid nodule +ve/ve Visual analogue scale HAQ score DAS28-CRP ESR (mm/h) CRP status +ve/ve RF status +ve/ve Anti-CCP status +ve/ve

M ± SD n (%)/n n (%)/n n (%)/n M ± SD M ± SD M ± SD n (%)/n n (%)/n M ± SD M ± SD M ± SD M ± SD n (%)/n n (%)/n n (%)/n

(%) (%) (%)

(%) (%)

(%) (%) (%)

Total

GG

GT+TT

p

37.1 ± 9.9 19 (17)/93 (83) 50 (44.6)/62 (55.4) 38 (33.9)/74 (66.1) 10.6 ± 6.6 8.9 ± 6.7 14.9 ± 8.9 19 (17)/93 (83) 46 (41.1)/66 (58.9) 6.0 ± 2.1 13.2 ± 6.5 4.6 ± 1.3 36.7 ± 21.4 77 (68.8)/35 (31.2) 83 (74.1)/29 (25.9) 104 (92.8)/8 (7.2)

37.1 ± 10.5 6 (11.1)/48 (88.9) 21 (38.9)/33 (61.1) 14 (25.9)/40 (74.1) 10.02 ± 6.8 8.4 ± 6.6 13.9 ± 9.2 9 (16.7)/45 (83.3) 25 (46.3)/29 (53.7) 5.8 ± 2.1 11.7 ± 5.8 4.4 ± 1.4 40.3 ± 23.9 36 (66.7)/18 (33.3) 41 (75.9)/13 (24.1) 52 (96.3)/2 (3.7)

37.0 ± 9.6 13 (22.4)/45 (77.6) 29 (50)/29 (50) 24 (41.4)/34 (58.6) 11.2 ± 6.4 9.4 ± 6.9 15.8 ± 8.7 10 (17.2)/48 (82.8) 21 (36.2)/37 (63.8) 6.0 ± 2.01 14.6 ± 6.8 4.6 ± 1.3 33.3 ± 18.3 41 (70.7)/17 (29.3) 42 (72.4)/16 (27.6) 52 (89.7)/6 (10.3)

0.95 0.13 0.26 0.11 0.36 0.45 0.28 1.0 0.34 0.37 0.02* 0.38 0.08 0.69 0.83 0.27

M ± SD = mean ± standard deviation; RF: rheumatoid factor; anti-CCP: anticyclic citrullinated peptide antibody; CRP: C-reactive protein; ESR: erythrocyte sedimentation rate; DAS28: RA disease activity score 28; VAS: visual analogue scale and HAQ: health assessment questionnaire. * p < 0.05 = significant.

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In conclusion, this study has confirmed the association between the STAT4 rs7574865 T allele with susceptibility and functional disability of RA. This might provide a good marker for diagnosis of disease susceptibility and severity with a potential benefit for developing pharmacotherapy targeting its expression in RA patients. Acknowledgment Authors are grateful for the Faculty members of Rheumatology Department, Mansoura University Hospital, Egypt for their help in selecting cases under the study. References [1] Firestein GS. Evolving concepts of rheumatoid arthritis. Nature 2003;423(6937):356–61. [2] Ikari K, Momohara S, Inoue E, Tomatsu T, Hara M, Yamanaka H, et al. Haplotype analysis revealed no association between the PTPN22 gene and RA in a Japanese population. Rheumatology (Oxford) 2006;45(11):1345–8. [3] Goëb V, Dieudé P, Daveau R, Thomas-L’otellier M, Jouen F, Hau F, et al. Contribution of PTPN22 1858T, TNFRII 196R and HLA-shared epitope alleles with rheumatoid factor and anti-citrullinated protein antibodies to very early rheumatoid arthritis diagnosis. Rheumatology (Oxford) 2008;47(8):1208–12. [4] Urayama KY, Thompson PD, Taylor M, Trachtenberg EA, Chokkalingam AP. Genetic variation in the extended major histocompatibility complex and susceptibility to childhood acute lymphoblastic leukemia: a review of the evidence. Front Oncol 2013;12(3):300. [5] Korman BD, Kastner DL, Gregersen PK, Remmers EF. STAT4: genetics, mechanisms, and implications for autoimmunity. Curr Allergy Asthma Rep 2008;8(5):398–403. [6] Frucht DM, Aringer M, Galon J, Danning C, Brown M, Fan S, et al. Stat4 is expressed in activated peripheral blood monocytes, dendritic cells, and macrophages at sites of Th1-mediated inflammation. J Immunol 2000;164(9):4659–64. [7] Mathur AN, Chang HC, Zisoulis DG, Stritesky GL, Yu Q, O’Malley JT, et al. Stat3 and Stat4 direct development of IL-17-secreting Th cells. J Immunol 2007;178(8):4901–7. [8] Watford WT, Hissong BD, Bream JH, Kanno Y, Muul L, O’Shea JJ. Signaling by IL12 and IL-23 and the immunoregulatory roles of STAT4. Immunol Rev 2004;202:139–56. [9] Zhao Y, Liu X, Liu X, Su Y, Li Y, Zhang X, et al. Association of STAT4 gene polymorphism with increased susceptibility of rheumatoid arthritis in a northern Chinese Han subpopulation. Int J Rheum Dis 2013;16(2):178–84. [10] Tong G, Zhang X, Tong W, Liu Y. Association between polymorphism in STAT4 gene and risk of rheumatoid arthritis: a meta-analysis. Hum Immunol 2013;74(5):586–92.

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