International Journal of

Molecular Sciences Article

RORC2 Genetic Variants and Serum Levels in Patients with Rheumatoid Arthritis Agnieszka Paradowska-Gorycka 1, *, Barbara Stypinska 1 , Andrzej Pawlik 2 , Katarzyna Romanowska-Prochnicka 3 , Ewa Haladyj 3 , Malgorzata Manczak 4 and Marzena Olesinska 3 1 2 3

4

*

Department of Biochemistry and Molecular Biology, National Institute of Geriatrics, Rheumatology and Rehabilitation, Spartanska ´ 1, 02-637 Warsaw, Poland; [email protected] Department of Pharmacology, Pomeranian Medical University, 70-111 Szczecin, Poland; [email protected] Department of Connective Tissue Diseases, National Institute of Geriatrics, Rheumatology and Rehabilitation, 02-637 Warsaw, Poland; [email protected] (K.R.-P.); [email protected] (E.H.); [email protected] (M.O.) Department of Epidemiology and Health Promotion, National Institute of Geriatrics, Rheumatology and Rehabilitation, 02-637 Warsaw, Poland; [email protected] Correspondence: [email protected]; Tel.: +48-22-844-5726

Academic Editor: Stephen A. Bustin Received: 15 February 2016; Accepted: 21 March 2016; Published: 1 April 2016

Abstract: Background: In the present study, we aimed to evaluate whether polymorphisms within the RORc2 gene are involved in the risk and severity of rheumatoid arthritis (RA). Methods: 591 RA patients and 341 healthy individuals were examined for RORc2 gene polymorphisms. Serum retinoic acid receptor-related orphan receptor C (RORc) levels were measured by enzyme-linked immunosorbent assay (ELISA). Results: The rs9826 A/G, rs12045886 T/C and rs9017 G/A RORc2 gene SNPs show no significant differences in the proportion of cases and control. Overall, rs9826 and rs9017 were in high linkage disequilibrium (LD) with D’ = 0.952 and r2 = 0.874, except rs9826 and rs12045886; and rs12045886 and rs9017 in weak LD. The genotype–phenotype analysis showed a significant association between RORc2 rs9826 A/G and rs9017 G/A single nucleotide polymorphisms (SNPs) and median of C-reactive protein (CRP). Serum RORc levels was higher in RA patients with rs9826AA, rs12045886TT and -TC, and rs9017AA genotypes compared to healthy subjects with the same genotypes (p = 0.02, p = 0.04 and p = 0.01, respectively). Moreover, the median of RORc protein level was higher in RA patients with number of swollen joints bigger then 3 (p = 0.04) and with Health Assessment Questionnaires (HAQ) score bigger then 1.5 (0.049). Conclusions: Current findings indicated that the RORc2 genetic polymorphism and the RORc2 protein level may be associated with severity of RA in the Polish population. Keywords: Th17 cells; rheumatoid arthritis; pathogenesis; RORc; polymorphisms

1. Introduction Since increasing interleukin (IL)-17 concentrations were observed in autoimmune diseases, increased attention has led to a precise definition of the role of human Th17 cells and their products in the process of ongoing immune response involved tissues [1–3]. The role of Th17 cells and Th17-associated cytokines in the pathogenesis of rheumatoid arthritis (RA) is now widely recognized. Th17 cells, which are the dominant effector T cell involved in the induction of autoimmune chronic tissue by promoting the immune response, are much more effective in inducing inflammation than any other Th1 cell recognized thus far and is therefore the “main culprit” of autoimmune diseases [4]. Th17 cell produce several cytokines including IL-17, IL-17F, IL-6, IL-21, IL-22 and tumor necrosis Int. J. Mol. Sci. 2016, 17, 488; doi:10.3390/ijms17040488

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factor-alpha (TNF-α) that play a key role not only in the RA but also in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and collagen induced arthritis (CIA), and they induces dendritic cells (DCs) to produce IL-12 and interferon (IFN-c) [5–10]. Th17 cell present in the joint may create a positive feedback loop leading to the continuous activation of T cells, which is a critical event in the generation of autoimmunity [11]. Although Th17 cells express numerous markers, human RORc2 (nuclear hormone retinoic acid receptor-related orphan receptor variant 2; a short isoform of RORc gene, encoded by the human RORc gene located on chromosome 1q21–q23) ortholog of mice RORγt, is a master transcriptional factor that can drive Th17 differentiation in both humans and mouse, respectively [12–16]. Overexpression of RORc2 by inducing IL-17, IL-26, TCR and CCR6 stimulates a wide range of phenotypic and functional programming during Th17 cells differentiation [17,18]. Knockdown of transcription factor RORc cause high Foxp3 levels and reduces expression of pro-inflammatory cytokines such as IL-1β, IL-6, IL-17, IFN-γ and TNF-α, suggesting that the role of RORc2 in Th17 cells differentiation involves not only in induction of Th17 characteristics genes, but also suppression of Treg cells specific programs [18–20]. The research of the Th17 cells development may help better understand its role in the pathogenesis of RA. However, there was still no too much study in this field. In this study, we hypothesized that the RORc2 gene is responsible for Th17 cells and IL-17-producing Treg cells differentiation may also be a strong molecular candidate for rheumatoid arthritis severity and/or susceptibility. To test this hypothesis, we examined, for the first time, three candidate single nucleotide polymorphisms (SNPs) in the RORc2 gene, rs9826 A/G, rs12045886 T/C and rs9017 G/A, and RORc protein expression and determined their possible association with susceptibility to and clinical phenotype of RA in Polish population. 2. Results 2.1. RORc2 SNPs Information and Association of the Individual SNPs with Risk of Rheumatoid Arthritis (RA) To confirm the genotyping results, PCR-amplified randomly selected DNA samples were analyzed on ABI PRISM Sequencer (Applied Biosystems, Foster City, CA, USA), and the results were 100% concordant (Figure 1). The RORc2 rs9826 A/G, rs12045886 T/C and rs9017 G/A polymorphism genotype distribution were in Hardy-Weinberg equilibrium (HWE) in both patients and control group (Table 1). Moreover, there was no evidence of any systematic bias in genotyping. The MAF of the three SNPs in our samples were similar to those in the Utah residents of northern and western European ancestry (HapMap database; Table 1). Table 1. SNPs information and genotyping results for rheumatoid arthritis (RA) patients and control group.

SNP ID rs9826 rs12045886 rs9017

Allele

SNP Type

A/G T/C G/A

31 UTR Intron 31 UTR

p (HWE)

MAF RA

Control

HapMap-CEU

RA

Control

0.38 0.27 0.64

0.34 0.25 0.64

0.39 0.33 0.60

0.12 0.18 0.14

0.55 0.53 0.95

MAF, minor allele frequency; HWE, Hardy-Weinberg equilibrium; CEU, Utah residents of northern and western European ancestry.

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Figure Sequencingmap mapofof genotype RORc2 gene. (A) rs9826 A/G, the of arrow of 1–3 Figure 1. 1. Sequencing genotype forfor RORc2 gene. (A) rs9826 A/G, the arrow 1–3 showed showed GG genotypes, respectively.; (B) rs12045886 theof arrow of 1–3 showed AA, AGAA, andAG GGand genotypes, respectively.; (B) rs12045886 T/C, theT/C, arrow 1–3 showed TT, TC TT, TT genotypes, respectively; (C) rs9017 arrow 1–3showed showedGG, GG, GA GA and AA andTCTTand genotypes, respectively; (C) rs9017 G/A,G/A, the the arrow of of 1–3 AA genotypes, respectively. genotypes, respectively.

Thegenotyping genotypingsuccess success was greater than incases. all cases. The distributions of genotype and The was greater than 87%87% in all The distributions of genotype and allele allele frequencies of the polymorphisms rs9826 A/G, rs12045886 T/C and rs9017 G/A in RORc2 among frequencies of the polymorphisms rs9826 A/G, rs12045886 T/C and rs9017 G/A in RORc2 among patientsand andcontrols, controls,as aswell wellas astheir theirassociations associationswith with the the risk riskof ofRA RAwere wereshown shownin inTable Table2.2.Three Three patients genetic models, including codominant, dominant and recessive were applied to assess the association genetic models, including codominant, dominant and recessive were applied to assess the association ofSNPs SNPswithin withinthe theRORc2 RORc2gene geneand andRA RArisk. risk.There There were significant differences in the proportion of were nono significant differences in the proportion of of cases and control under each genetic model for tested polymorphisms. Effect sizes were adjusted cases and control under each genetic model for tested polymorphisms. Effect sizes were adjusted for for and sex and age and results still insignificant. sex age and results werewere still insignificant. Table Haplotype 2. Genotype and allele of the RORc2 polymorphisms in RA patients and controls. 2.2. RORc2 Analysis andfrequencies Risk of Rheumatoid Arthritis RA Controls Unadjusted OR gene Adjusted OR and their p polymorphisms p Next, we evaluated the interaction between examined RORc2 Genotype n (%) n (%) (95% CI) Value (95% CI) Value inheritance by analyzing the distribution of haplotypes in RA patients and control group. The (A/G) interaction between any possible pair of SNPsrs9826 was visualized by SHEsis program (Figure 2). Analysis AA 221 (50%) 144 (43%) 1 – – revealed high linkage disequilibrium (LD) between rs9826 and rs9017 (D’ = 0.9521 and r2 = 0.874), Codominant AG 295 (37%) 157 (46%) 1.04 (0.86–1.27) 0.68 1.14 (0.83–1.55) 2 0.42 but weak LD between rs9826 and rs12045886; and rs12045886 and rs9017 (D’ = 0.309 and r = 0.059; GG 75 (13%) 37 (11%) 1.13 (0.85–1.49) 0.41 1.03 (0.66–1.60) 0.89 D’ = 0.345 and r2 = 0.070, respectively). The eight potential haplotypes were formed, as present in AA 221 (50%) 144 (43%) 1 – 1 – Dominant Table 3. Four major haplotypes (ACA, and GTG), frequency >5%,0.23 were AG +RORc2 GG 370 (63%) 194 (57%) ATA, 1.12GCG, (0.97–1.28) 0.12with1.14 (0.92–1.43) observed in RA patients in healthy subjects. Among AA + AGas well 516 as (87%) 301 (89%) 1 them, the–most frequent 1 haplotype–was Recessive ATA, which was estimate on75 589 of the 1186 chromosome with a frequency of 49.6% in RA patients GG (13%) 37 (11%) 1.09 (0.88–1.34) 0.43 1.01 (0.73–1.41) 0.93 RORc2 SNP

Codominant

TT

281 (53%)

rs12045886 (T/C) 190 (56%) 1

–

1

–

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and on 367 of the 682 chromosomes with frequency of 53.8% in control group. This ATA haplotype demonstrated significantly lower frequencies in RA patients compared to controls (p = 0.04, odds ratio (OR) = 0.820, confidence interval (CI) = 0.677–0.994). This association is translated into a protective effect. There were no differences in haplotype frequency between RA patients and control group for the other haplotypes. Table 2. Genotype and allele frequencies of the RORc2 polymorphisms in RA patients and controls. RORc2 SNP

Genotype

RA n (%)

Controls n (%)

Codominant

AA AG GG

221 (50%) 295 (37%) 75 (13%)

144 (43%) 157 (46%) 37 (11%)

Dominant

AA AG + GG

221 (50%) 370 (63%)

Recessive

AA + AG GG

p Value

Adjusted OR (95% CI)

p Value

1 1.04 (0.86–1.27) 1.13 (0.85–1.49)

– 0.68 0.41

1 1.14 (0.83–1.55) 1.03 (0.66–1.60)

– 0.42 0.89

144 (43%) 194 (57%)

1 1.12 (0.97–1.28)

– 0.12

1 1.14 (0.92–1.43)

– 0.23

516 (87%) 75 (13%)

301 (89%) 37 (11%)

1 1.09 (0.88–1.34)

– 0.43

1 1.01 (0.73–1.41)

– 0.93

Codominant

TT TC CC

281 (53%) 221 (41%) 32 (6%)

190 (56%) 132 (39%) 19 (6%)

1 1.04 (0.81–1.33) 1.05 (0.71–1.55)

– 0.75 0.82

1 1.35 (0.92–1.98) 1.61 (0.34–1.10)

– 0.12 0.10

Dominant

TT TC + CC

281 (53%) 253 (47%)

190 (56%) 151 (44%)

1 1.06 (0.93–1.22)

– 0.37

1 1.00 (0.80–1.24)

– 0.98

Recessive

TT + TC CC

502 (94%) 32 (6%)

322 (94%) 19 (6%)

1 1.04 (0.78–1.39)

– 0.80

1 0.69 (0.44–1.08)

– 0.10

Unadjusted OR (95% CI)

rs9826 (A/G)

rs12045886 (T/C)

rs9017 (G/A) Codominant

GG GA AA

67 (13%) 262 (50%) 194 (37%)

43 (13%) 157 (46%) 141 (41%)

1 1.09 (0.90–1.33) 0.90 (0.73–1.10)

– 0.39 0.31

1 1.17 (0.86–1.60) 0.94 (0.68–1.30)

– 0.31 0.72

Dominant

GG GA + AA

67 (13%) 456 (87%)

43 (13%) 298 (87%)

1 0.99 (0.81–1.22)

– 0.93

1 1.09 (0.79–1.49)

– 0.61

Recessive

GG + GA AA

329 (63%) 194 (37%)

200 (59%) 141 (41%)

1 0.92 (0.80–1.05)

– 0.21

1 0.92 (0.74–1.15)

– 0.47

OR, odds ratio; CI, confidence interval; Adjusted OR for sex and age.

Table 3. RORc2 haplotypes in RA patients and controls. Haplotype rs9826/rs12045886/rs9017

RA n = 1186 (%)

Controls n = 682 (%)

OR (95% CI)

p Value

ACA ACG ATA ATG GCA GCG GTA GTG

133 (11.2) 9 (0.8) 589 (49.5) 11 (0.9) 2 (0.1) 172 (14.5) 9 (0.8) 261 (22.2)

63 (9.2) 8 (1.2) 367 (53.8) 8 (1.2) 3 (0.4) 97 (14.2) 6 (0.9) 130 (19.1)

1.235 (0.900–1.695) – 0.820 (0.677–0.994) – – 1.016 (0.776–1.331) – 1.178 (0.930–1.492)

0.19 – 0.04 – – 0.91 – 0.17

p, Fisher’s test; p considered as significant was bold.

was ATA, which was estimate on 589 of the 1186 chromosome with a frequency of 49.6% in RA patients and on 367 of the 682 chromosomes with frequency of 53.8% in control group. This ATA haplotype demonstrated significantly lower frequencies in RA patients compared to controls (p = 0.04, odds ratio (OR) = 0.820, confidence interval (CI) = 0.677–0.994). This association is translated intoJ. Mol. a protective effect. and Int. Sci. 2016, 17, 488 There were no differences in haplotype frequency between RA patients 5 of 15 control group for the other haplotypes.

Figure 2. Linkage disequilibrium (LD) plots of three SNPs in the RORc2 gene. The plot illustrates Figure 2. Linkage disequilibrium (LD) plots of three SNPs in the RORc2 gene. The plot illustrates pairwiseLD LDbetween between polymorphisms based D′ values. approaching zero indicate 1 values. pairwise allall polymorphisms based on Don ValuesValues approaching zero indicate absence absence of LD, and those approaching 100 indicate complete LD. The square colored red of LD, and those approaching 100 indicate complete LD. The square colored red representrepresent varying varying of degrees LDLOD < 1 and LOD (logarithm 2 scores (strong LD) and white blocks degrees LD < 1ofand (logarithm of odds) >of2odds) scores>(strong LD) and white blocks represent represent varying of LD < 1