Lack of association between STAT4 rs7574865 polymorphism and autoimmune diseases including rheumatoid arthritis and systemic sclerosis in Southwest Iran

Accepted Manuscript Lack of association between STAT4 rs7574865 polymorphism and autoimmune diseases including rheumatoid arthritis and systemic sclerosis in Southwest Iran

Nazanin Nezaratian, Seyed Reza Kazemi Nezhad, Mohammdreza Hajjari, Mohammd Reza Akhoond PII: DOI: Reference:

S2214-5400(17)30064-6 doi: 10.1016/j.mgene.2017.08.002 MGENE 352

To appear in:

Meta Gene

Received date: Revised date: Accepted date:

27 April 2017 28 July 2017 3 August 2017

Please cite this article as: Nazanin Nezaratian, Seyed Reza Kazemi Nezhad, Mohammdreza Hajjari, Mohammd Reza Akhoond , Lack of association between STAT4 rs7574865 polymorphism and autoimmune diseases including rheumatoid arthritis and systemic sclerosis in Southwest Iran, Meta Gene (2017), doi: 10.1016/ j.mgene.2017.08.002

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ACCEPTED MANUSCRIPT Research Article

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Lack of Association between STAT4 rs7574865 Polymorphism and Autoimmune Diseases Including Rheumatoid Arthritis and Systemic Sclerosis in Southwest Iran

Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran

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Nazanin Nezaratiana, Seyed Reza Kazemi Nezhada,*, Mohammdreza Hajjaria, Mohammd Reza Akhoondb

Department of Statistics, Faculty of Mathematics and Computer Science, Shahid Chamran

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University of Ahvaz, Ahvaz, Iran *

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Corresponding author: Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran. Tel. & Fax: +98-6133338965, E-mail: [email protected], [email protected]

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Running Title: Lack of Association between STAT4 with RA and SSc.

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ACCEPTED MANUSCRIPT Abstract

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Systemic sclerosis (SSc) and Rheumatoid arthritis (RA) are types of autoimmune diseases. Susceptibility to these diseases depends on a complex interaction between environment and genetic factors. It is likely that common underlying genes are involved in mentioned diseases. In recent years, researchers focused on the genes encoding immune regulatory proteins, such as the STAT4 that encodes a transcription factor and plays a key role in the signaling pathways of IL12, IL-23 and type 1 interferon. Several reports have demonstrated that these pathways are involved in RA and SSc pathogenesis. Single nucleotide polymorphism (SNP)" rs7574865 G>T" in the STAT4 gene, has been reported to be associated with RA and SSc in many ethnic groups. The aim of the present study was to investigate the association of rs7574865 polymorphism in STAT4 gene with RA and SSc diseases in Khuzestan province of Iran. The study included 320 persons (120 patients with RA and 80 patients with SSc and 120 matched healthy controls). Genomic DNA was isolated and genotyped using Restriction Fragment Length Polymorphismpolymerase chain reaction (PCR-RFLP) assay for the STAT4 gene rs7574865 polymorphism. The statistical analyses were performed by SPSS software. No significant association was observed between STAT4 rs7574865 polymorphism and RA (P value = 0.916) as well as SSc (P value = 0.344) in the Khuzestan province population. The correlation was examined between the clinical characteristics (RF and anti-CCP) and genotype frequencies. No evidence was found between anti-CCP and STAT4 rs7574865 genotypes. However, our result showed significant association between STAT4 rs7574865 genotypes and RF in this population (p = 0.01). Our findings suggest that there is no association between STAT4 rs7574865 polymorphism with susceptibility to RA and SSc in Khuzestan province.

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Keywords: Anti-CCP, Polymorphism, Rheumatoid factor, Rheumatoid arthritis, STAT4, Systemic sclerosis.

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ACCEPTED MANUSCRIPT 1. Introduction Autoimmune diseases are a diverse group of complex disorders characterized by the development of immune responses against self-antigens and affect about 5% of the population worldwide (Gregersen and Olsson, 2009; Marrack et al., 2001; Simmonds and Gough, 2004, Wandstrat and Wakeland, 2001). Autoimmune diseases are caused by an interaction of genetic and environmental factors and share a number of characteristic that suggest common pathogenesis pathways (Becker, 2004; Tait et al., 2004). Systemic sclerosis (SSc) and Rheumatoid arthritis (RA) are types of autoimmune diseases which have

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overlapping genetic susceptibility loci (Dieude et al., 2009). Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that is associated with progressive

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disability and systemic complications. RA is characterized by synovial inflammation, cartilage and bone

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destruction and autoantibody production (rheumatoid factor and anti-citrullinated protein antibody) (Aletaha et al., 2010; McInnes and Schett, 2011). Its prevalence is approximately 0.5-1%worldwide. Twin

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studies implicate genetic factors in RA, with concordance rate of 15 to 30% among monozygotic twins and 5% among dizygotic twins (Begovich et al., 2004; MacGregor et al., 2000).

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Systemic sclerosis is a multisystem and connective tissue disorder characterized by fibrosis in skin and internal organ and immune dysregulation (Martin and Fonseca, 2011). Fibrosis can caused by profibrotic cytokines, such as TGF beta, IL-4, and PDGF (Sakkas, 2005). In this disease circulating autoantibodies,

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altered immune mediators, and infiltration of mononuclear cells in affected organ argue that immune

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system dysfunction drives pathogenesis (Gu et al., 2008). SSc is subdivided into limited cutaneous SSc (lcSSc) and diffuse cutaneous (dcSSc) types based on the extent of skin involvement (Ramos et al., 2015). Nearly 70% of SSc patients show pulmonary involvement, which is the main cause of mortality in

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SSc (Steen and Medsger, 2007).

RA and SSc are very heterogeneous (Ramos et al., 2015). The incidence of RA is three times higher in

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women than men, also SSc is associated with a strong sex bias as well as a female predominance was reported with between four and nine affected women for one man (Ferri et al., 2002; Meier et al., 2012). There have been numerous studies that identified several robust RA and SSc susceptibility loci, such as STAT4 gene that its association has been firmly confirmed with both diseases. Signal transducer and activators of transcription 4 (STAT4) encodes a transcription factor which is expressed in activated peripheral blood monocytes, dendritic cells and macrophages at the sites of inflammation (Settin et al., 2014) . It induces the signals of several cytokines, such as IL-12, IL-23, IL-27 and type I interferons (IFNs). STAT4 mediates signals into Th1 and Th17 differentiation, monocyte activation and interferon-gamma production (Ciric et al., 2009; Watford et al., 2004). STAT4 is localized in the cytoplasm and can be phosphorylated by membrane-bound receptor, dimerized and transferred to 3

ACCEPTED MANUSCRIPT the nucleus. As a result, STAT4 dimers bind to specific sequences of cytokine-inducible genes and activate gene transcription (Horvath, 2000). There are several SNPs in this gene. The STAT4 rs7574865 polymorphism is located within the third intron of the gene and shows the strongest association with autoimmune diseases including RA, SSc, systemic lupus erythematosus (SLE), Behcet's disease and type 1 diabetes in many ethnic groups (Gu et al., 2015; Korman et al., 2008; Liang et al., 2012; Yi et al., 2013). However, there have been some conflicting results reporting no association between RA/SSc and STAT4 rs7574865 polymorphism (Plant et al., 2010; Yuksel, 2014). Also an association study that included some independent populations of Caucasian ancestry demonstrated that STAT4 rs7574865

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polymorphism confers susceptibility to lcSSc, but not to dcSSc (Rueda et al., 2009).

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2. Objectives

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In present study, we investigated the association between STAT4 rs7574865 polymorphism and

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susceptibility to development of RA and SSc in the Khuzestan province population.

3. Materials and Methods

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In this study, all patients and subjects of the control group were chosen among a sample of the population of Khuzestan province in the southwest of Iran. After obtaining the participants informed consent and receiving the approval of the ethics committee of Ahvaz Jundishapur University of Medical Sciences, 120

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RA patients (99 female, 21 male) with the average age of 44.83 years and 80 SSc patients (67 female, 13

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male) with the average age of 44.3 years and age range of 30-75 years and 120 matched healthy controls (95 female, 25 male) with the average age of 46.93 years were consecutively recruited from Rheumatology Clinic at Golestan Hospital in Ahvaz, Iran. All patients included in the study fulfilled the

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American College of Rheumatology (ACR) 1987 revised criteria for RA and SSc. None of the healthy controls had a family history of autoimmune diseases. Rheumatoid factor (RF) measured to be >20

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IU/mL was considered as RF positive. The presence of anti-cyclic citrullinated peptide (anti-CCP) antibodies was determined and a cut-off point of >5 U/mL was used as a stringent criterion for anti-CCP positive.

3.1. DNA genotyping Five microliters blood was collected from all subjects and stored in -40°C. Then DNA was extracted using salting-out method and was checked out by Nanodrop 1000 spectrophotometer and agarose electrophoresis. For genotyping of rs7574865, we employed Restriction Fragment Length Polymorphismpolymerase chain

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(PCR-RFLP) technique.

The primers

that were used

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AAAGAAGTGGGATAAAAAGAAGTTTG-3' and 5'-CCACTGAAATAAGATAACCACTGT-3'. The 25 µL PCR reactions contained 2 µL template DNA (~100 ng µLˉ1), 1 µL of each primer (10 µM), 12.5 4

ACCEPTED MANUSCRIPT µL of PCR Master Mix, and 8.5 µL sterile distilled water. Thermal cycling was achieved through the initial activation step at 95°C for 3 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 45 s, extension at 72°C for 45 s and a final extension at 72°C for 5 min .The 147 bp PCR products were digested with Hpa1 enzyme (Thermo Scientific) at 37°C for 16 h. The Hpa1 enzyme had one restriction site in the amplicon. The G allele was not digested so the GG genotype appeared as a single band developed at 147 bp while the T allele was cut into 122 and 25 bp fragments, so the TT genotype appeared as single band developed at 122 bp and the GT genotype appeared as two bands. For confirming PCR-RFLP quality, some genotyping results were validated by direct sequencing method and no

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genotyping mistake was found. 3.2. Statistical analysis

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Data analysis was performed with the statistical program SPSS (SPSS V18, SPSS Inc, Chicago, IL,

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USA). Comparison of continuous variables, such age and biochemical characteristics between all RA and SSc patients and control groups were done by independent samples T test or Mann-Whitney test. To

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estimate the risk of the disease, thr logistic regression model was used to calculate odds ratio (OR) with

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95% confidence intervals (CIs). P value of <0.05 was considered as statistically significant.

4. Results

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4.1. Correlation between clinical features of the RA/SSc patients and STAT4 gene rs7574865

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polymorphism

The demographic and clinical data of the RA and SSc patients are shown in Table 1. Agarose gel electrophoresis regards to RFLP is shown in Figure 1. There was no significant genotypic association

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with age, race and sex ( p > 0.05) (data are not shown). Of the 120 RA patients tested for anti-CCP antibodies, 45.8% were positives and 54.2% were negative for these antibodies. RA patients were classified according to anti-CCP status, no meaningful differences were observed between positive

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patients compared with negative patients (p = 0.098). As it was shown in table 4, anti-CCP positive and negative patients were survey regard to GG, GT and TT genotypes as well as G and T allele distributions and no association was calculated. The RA patients were stratified into RF positive and negative groups, as well. Also, 32.5% of RA patients were positives and 67.5% were negative for RF. The significant differences was observed in genotype distributions in positive patients compared with negative patients (p = 0.01). This two groups including positive and negative RF patients were analysed regarding to genotypes and alleles were shown in table 5. None of them showed correlation excepts for G allele (Table 5). A similar result was also found between SSc patients and control subjects. The SSc patients were divided into limited SSc and 5

ACCEPTED MANUSCRIPT diffuse SSc. We found no statistically significant association between both groups (p = 0.694) and neither GG, GT, TT genotypes or G and T alleles (Table 6) . 4.2. Genotype and allele frequencies of the rs7574865 STAT4 gene polymorphism in RA and SSc patients The genotypic and allelic frequencies of the rs7574865 STAT4 gene SNP in RA, SSc patients as well as control subjects are shown in Tables 2 and 3. Genotype frequencies for the STAT4 rs7574865 SNP were in Hardy-Weinberg equilibrium in both patients and control cohorts. There was no statistically significant differences between RA patients and controls (p = 0.916), also no significant association was observed

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between SSc patients and control groups (p = 0.344). The frequency of the minor T allele was also very similar in RA patients (49.6%) and healthy controls (50.3%).

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Our results demonstrated that the STAT4 rs7574865 polymorphism is not associated with RA and SSc in

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Khuzestan province, Southwest Iran.

5. Discussion

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The STAT4 is an essential transcription factor for regulation of the immune responses. The STAT4 plays a key role in the differentiation of Th1 and Th17 cells that are implicated in RA and SSc pathogenesis (Frucht et al., 2000; Watford et al., 2004). The four single nucleotide polymorphisms (SNPs) including

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rs18889341, rs7574865, rs8179673 and rs10181656 are in the third intron of the STAT4 gene and they

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formed a susceptibility haplotype in RA that were replicated and confirmed in different ethnic populations. Among these, rs7574865 SNP had the most significant association with RA and SSc in many ethnic groups (Korman et al., 2008). One meta-analysis study including 28 comparisons which involved

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19 European, 5 Asian, 3 African and one Latin American populations showed an association between STAT4 rs7574865 SNP and RA (Gu et al., 2015). Moreover, some studies were conducted on the Han population of Southwest china (Li et al., 2009), Turkish (Yuksel, 2014), African American (Kelley et al.,

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2010) and European populations showed no significant association between this SNP and RA (Plant et al., 2010). Many studies demonstrated meaningful association of the STAT4 rs7574865 and SSc susceptibility in Japanese, Chinese as well as Caucasians of North American, Spanish and European ancestry (Yi et al., 2013). In the present study, no positive association was observed between STAT4 rs7574865 SNP neither with RA nor with SSc in the Khuzestan province population Although functional consequence of rs7574865 SNP remains unclear, it may influence on STAT4 expression at transcription or splicing variation levels. A recent study demonstrated a significant association of the STAT4 risk allele with STAT4 overexpression in osteoblasts but not in B cells. This

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ACCEPTED MANUSCRIPT might indicate the potential presence of a tissue-specific intragenic enhancer and cell type specific effects of different STAT4 gene variants on STAT4 expression levels (Sigurdsson et al., 2008). In a recent study that was carried out on Iranian population, no significant correlations was reported between Juvenile rheumatoid arthritis (JRA) as an autoimmune disease with non of the STAT4 rs7574865 SNPs (Aslani et al., 2017). Another survey showed a significant association between rs7574865 T allele and susceptibility to Systemic lupus Erythematosus (SLE). The rs7574865 TT and GT genotypes demonstrated a significant association with the risk of SLE in the Iranian population and it was found that

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STAT4 rs7574865 is associated with SLE (Mirkazemi et al., 2013) although no association was observed between STAT4 rs7574865 polymorphisms with RA and SSc in this study.

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A survey on Mexican population reported that STAT4 rs7574865 G/T polymorphism is associated with

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RA and disease activity, but not with anti-CCP antibody levels (Durán-Avelar et al., 2016) as we obtained the same results in this study. Furthermore, it was confirmed that a significantly increased frequency of the minor T allele in RA patients compared with healthy controls in the Spanish, the Dutch and

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Swedish populations and no more correlation between rs7574865 and the presence of RF or anti-CCP autoantibodies was found (Orozco G et al., 2008) and the results are similar to this study. On the contrary

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to current study, Egyptian RA patients with the TT genotype had significantly higher levels of the antiCCP, compared to those with GG and GT genotypes (El-Saadany, et al, 2016).

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RA and SSc are multifactorial disorders and several genes are involved in the pathogenesis of these diseases. Therefore, it is possible that polymorphisms in other genes associated with susceptibility of RA

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and SSs in this population. Moreover, the possibility that the other polymorphisms or mutations in STAT4 gene represent a risk factor for development of RA and SSc this population cannot be ruled out. The differences observed between our findings and other populations could be due to discrepancy in the

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genetic background of the populations, sample size of studies and environmental factors effect like lifestyles. Small sample size may have an effect on the statistic power. Therefore, our results need

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confirmation in larger sample size assays. On the other hand, population of Khuzestan province is heterogeneous and consists of Arab, Lor, Kord and Persian ethnicities. It might be another reason for the lack of association between STAT4 gene rs7574865 polymorphism with RA and SSs diseases in population of Khuzestan province. In conclusion, this study gives further support to the importance of replication studies as susceptible loci that might be differ in various ethnic groups

Authors' contribution

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ACCEPTED MANUSCRIPT Study concept and design: Seyed Reza Kazemi Nezhad. Interpretation of findings: Nazanin Nezaratian, Seyed Reza Kazemi Nezhad, Mohammdreza Hajjari. Drafting of the manuscript: Nazanin Nezaratian. Critical revision of the manuscript for important intellectual content: Nazanin Nezaratian, Seyed Reza Kazemi Nezhad, Mohammdreza Hajjari. Statistical analysis: Mohammd Reza Akhoond

funding/support

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This study as an academic scholarly work was supported by the Master's thesis grant from Shahid Chamran University of Ahvaz.

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Acknowledgments This project was supported by Master's thesis grant from Shahid Chamran University of Ahvaz, Khuzestan (Iran). The authors wish to appreciate Rheumatology Clinic at Golestan Hospital of Ahvaz for providing blood samples of patient individuals.

Conflicts of Interest

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The authors declare that there is no conflict of interests regarding the publication of this paper.

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Watford, W.T., Hissong, B.D., Bream, J.H., Kanno, Y., Muul, L., O'Shea, J.J., 2004. Signaling by IL-12 and IL-23 and the immunoregulatory roles of STAT4. Immunol. Rev. 202, 139-156. Yi, L., Wang., J.C., Guo, X.J., Gu, Y.H., Tu, WZ., Guo, G., Yang, L., Xiao, R., Yu, L., Mayes, M.D., Assassi, S., 2013. STAT4 is a genetic risk factor for systemic sclerosis in a Chinese population. Int. J. Immunopathol. Pharmacol. 26, 473-478. Yuksel, B., Ataman, S., Evcik, D., Ay, S., Mumcuoglu, M., Erdogan, B., 2014. Is there an association between two-STAT4 gene polymorphisms and rheumatoid arthritis in Turkish population? Arch. Rheumatol. 29, 20-28.

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ACCEPTED MANUSCRIPT Table 1. Demographic and clinical characteristics of the RA and SSc patients. RA patients

Demographic

SSc patients

Controls

(n=80)

(n=120)

Age (years)

44.83±11.87

44.3±11.34

46.93±11.89

Gender (females/males)

99/21 (82.5/17.5%)

67/13 (63.7/16.3%)

95/25 (79.2/20.8%)

Clinical 7.15±6.23

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Anti-CCP Ab positive (Anti-CCP Ab positive/total patients)

79/120 (66%)

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Anti-CCP Ab negative (Anti-CCP Ab negative /total patients)

41/120 (34%)

RF positive (RF positive/total patients)

42/120 (35%)

RF negative (RF negative /total patients)

78/120 (65%)

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Disease duration (years)

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Abbreviation: Anti-CCP, anti-cyclic citrullinated peptide; Ab, antibody; RF, rheumatoid factor; RA, Rheumatoid Arthritis; SSc, Systemic Sclerosis.

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ACCEPTED MANUSCRIPT Table 2. Distribution of the STAT4 rs7574865 polymorphism frequency in RA patients and controls. RA patients (n=120)

Controls (n=120)

p-value

OR

95% CI

GG

58

48.3%

56

46.7%

-

-

-

GT

53

44.2%

56

46.7%

0.737

0.914

0.540-1/545

TT

9

7.5%

8

6.7%

0.874

1.086

0.391-3.014

G

169

50.1%

168

49.8%

0.921

T

71

49.6%

72

50.3%

-

Genotype

PT

Allele 0.690-1.509

-

-

RI

1.020

AC

CE

PT E

D

MA

NU

SC

The values are presented as frequency in percentage and number of genotypes or alleles. The frequencies comparison between groups was analyzed by Chi-Square test. Statistical significance was at p < 0.05. RA, rheumatoid arthritis; OR, odds ratio; CI, confidence interval.

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ACCEPTED MANUSCRIPT Table 3. Distribution of the STAT4 rs7574865 polymorphism frequency in SSc patients and controls. SSc patients (n=120)

Controls (n=120)

p-value

OR

95% CI

Genotype GG

33

41.2%

56

GT

37

46.2%

56

TT

10

12.5%

8

46.6% 46.6% 6.7%

-

-

-

0.758

2.121

0.617-2.039

0.150

1.121

0.762-5.908

103

38.0%

168

T

57

44.1%

72

62%

0.239

55.8%

-

-

0.774

0.506-1.185

-

-

RI

G

PT

Allele

AC

CE

PT E

D

MA

NU

SC

values are presented as frequency in percentage, number of genotypes or alleles data. The frequencies comparison between groups was analyzed by Chi-Square test. Statistical significance was at p < 0.05.SSc, systemic sclerosis; OR, odds ratio; CI, confidence interval.

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ACCEPTED MANUSCRIPT Table 4. Comparison of STAT4 rs7574865 genotypes in RA patients stratified according to anti-CCP antibody anti-CCP antibody positive

anti-CCP antibody

p-value

OR

95% CI

negative

Genotype 39

67.2%

19

GT

6

66.7%

3

TT

37

69.8%

16

G

115

68.0%

54

T

43

60.0%

28

32.8% 33.3%

-

-

-

0.771

1.127

0.505-2.514

0.63

0.244

0.055-1.081

1.387

0.780-2.465

-

-

30.2%

Allele 0.265

39.4%

SC

-

RI

31.9%

PT

GG

AC

CE

PT E

D

MA

NU

Referred as subgroups of rheumatoid arthritis patients; OR, odds ratio; CI, confidence interval; anti-CCP, anti-cyclic citrullinated peptide

15

ACCEPTED MANUSCRIPT Table 5. Comparison of STAT4 rs7574865 genotypes in RA patients stratified according to RF antibody.

RF antibody positive

RF antibody negative

p-value

OR

95% CI

Genotype GG

27

46.6%

31

53.4%

-

-

-

GT TT

15 0

28.3% 0%

38 9

71.7% 100%

0.052 0.057

0.461 0.60

0.211-1.007 0.003-1.084

G

69

40.8%

100

59.2%

0.004

2.576

1.349-4.920

T

15

21.1%

56

78.8%

-

PT

Allele

-

-

AC

CE

PT E

D

MA

NU

SC

RI

Referred as subgroups of rheumatoid arthritis patients; OR, odds ratio; CI, confidence interval; RF, rheumatoid factor.

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ACCEPTED MANUSCRIPT Table 6. Comparison of STAT4 rs7574865 genotypes in SSc patients stratified into lcSSc and dcSSc types. lcSSc

p-value

dcSSc

OR

95% CI

Genotype GG

11

33.3%

22

66.7%

-

-

-

GT TT

16 4

43.2% 40.0%

21 6

56.8% 60.0%

0.396 0.699

0.656 0.750

0.248-1.736 0.175-3.222

G

38

36.9%

65

63.1%

0.517

1.244

0.642-2.409

T

24

42.1%

33

57.9%

-

PT

Allele

-

-

RI

Referred as subgroups of systemic sclerosis patients; OR, odds ratio; CI, confidence interval; lcSSc, limited cutaneous SSc,

AC

CE

PT E

D

MA

NU

SC

dcSSc, diffuse cutaneous SSc.

17

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

Figure 1. STAT4 rs7574865 gene polymorphism agarose gel electrophoresis image after RFLP. column L100 is 100 bp marker, columns 1 and 3 are GG homozygote samples develops at 147 bp, columns 2, 4 and 5 are GT heterozygote samples and column 6 and 7 are TT mutated homozygote genotypes that appear at 122 bp.

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ACCEPTED MANUSCRIPT Our Research Highlights: This is the first study that analyzes the association of the rs7574865 G/T STAT4 SNP with RA and SSc in Iranian population.



Rs7574865 G/T STAT4 SNP is not associated with RA in Khuzestan province population, southwest Iran.



Rs7574865 G/T STAT4 SNP is not associated with a susceptibility to SSc in the Khuzestan province population.



The differences observed between our finding and other populations could be due to

PT



discrepancy in the genetic background of the populations, sample size of studies and

The population of Khuzestan province is heterogeneous and consists of several race. It

SC

might be another reason for the lack of association between STAT4 gene rs7574865

CE

PT E

D

MA

NU

polymorphism with RA and SSs diseases in population of Khuzestan province.

AC



RI

environmental effect, like life-style.

19