TY - JOUR
AU - Olesinska, M
AB - Summary One among many factors involved in induction of rheumatoid arthritis (RA) are T cells, the differentiation of which depends upon a unique combination of stimulants and subsequent activation of diverse transcription factors. The aim of this study was to identify polymorphic variants in Smad3 and NFATc2 genes and their possible association with susceptibility to and severity of RA. A total of 272 RA patients, 321 for Smad3 and 304 for nuclear factor of activated T cells (NFAT)c2 healthy individuals, were examined for rs6494629 C/T and rs2289263 T/G Smad3 and rs880324 NFATc2 gene polymorphisms using the polymerase chain reaction–fragment length polymorphism (PCR–RFLP) method and TaqMan single nucleotide polymorphism (SNP) genotyping assay, respectively. Serum Smad3 and NFATc2 levels in RA patients and controls were measured by enzyme-linked immunosorbent assay (ELISA). The rs6494629 C/T Smad3 gene polymorphism under the recessive (TT versus CC+CT) and over-dominant (CC+TT versus CT) models were associated with RA (P = 0·014 and P = 0·008, respectively). Smad3 rs2289263 T/G revealed differences in the case–control distribution in co-dominant, recessive and over-dominant models (P = 0·037, P = 0·010, P = 0·034). Overall, rs6494629 C/T and rs2289263 T/G Smad3 gene polymorphisms were in a weak linkage disequilibrium (LD) with D′ = 0·116 and r2 = 0·004. After Bonferroni correction, the genotype–phenotype analysis showed no significant correlation of the Smad3 rs6494629 C/T and rs2289263 T/G and NFATc2 rs2289263 TT polymorphisms with disease activity, joint damage and extra-articular manifestation in RA patients. Serum Smad3 and NFATc2 levels were significantly higher in RA patients than in control groups (both P = 0 0000). The present findings indicated that Smad3 genetic polymorphisms may be associated with the susceptibility to RA in the Polish population. NFATc2, polymorphisms, rheumatoid arthritis, serum levels, Smad3 Introduction Rheumatoid arthritis (RA) is one of the chronic autoimmune diseases, with genetic and environmental predisposition [1–4]. The genetic associations of the human leucocyte antigen (HLA) shared epitope alleles with developing of RA indicate that the disease is driven at least partially by T cells that could be targeted in clinical therapy [5]. Recent studies have shown that in T cells, nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 2 (NFATc2) NFAT1 or NFATp) and SMAD family member 3 (Smad3) are important factors regulating T cell activation and production of inflammatory cytokines [6–11]. Both these transcriptional factors regulate the activity of numerous gene expressions by themselves, but can also co-operate with other transcriptional factors at composite sites within the promoters of target genes [6,12,13]. Moreover, both are needed for the activity of forkhead box protein 3 (FoxP3) gene enhancer and induction of FoxP3 [12,14]. NFATc2 is a main transcriptional factor that highly up-regulates gene expressions such as proteinases, cyclo-oxygenase type 2 (Cox-2) and proinflammatory cytokines [e.g. interleukin (IL)-2, IL-6, IL-12, tumour necrosis factor (TNF)-α], which may cause dysfunction of articular chondrocytes, differentiation of osteoclast, progressive loss of articular cartilage and cartilage proliferation in joints [15–18]. Furthermore, the Smad3 signalling pathway is essential for maintaining articular cartilage by inhibiting terminal hypertrophic differentiation of chondrocytes and regulating synthesis of matrix components [19,20]. In the present study we examined two candidate single nucleotide polymorphisms (SNPs) in the Smad3 gene: rs6494629 C/T and rs2289263 T/G, and one SNP in the NFATc2: rs880324 G/A, and explored their association with susceptibility to and severity of RA in the Polish population. Materials and methods Study population In total, 272 patients with established RA and 321 for Smad3 and 304 for NFATc2 unrelated healthy controls were included in this study. All patients fulfilled the American College of Rheumatology (ACR 1987) criteria for RA. Patients with RA were recruited from the Connective Tissue Diseases Department of the Institute of Rheumatology in Warsaw. All patients signed an informed consent and clinical and biochemical data were collected from patients' files and questionnaires and summarized in Table 1. Table 1 Baseline clinical and demographic features of the study patients with rheumatoid arthritis (RA). Data are expressed as median with interquartile ranges (IQR) or percentages . RA patients . Characteristics . N* . Median (IQR) . Age (years) 268 56 (50–64) Disease duration (years) 265 10 (6–17) Larsen–Dale index 271 3 (3–4) Number of swollen joints 266 3 (1–7) Number of tender joints 266 8 (4–13) ESR (mm/h) 270 28 (15–42) CRP (mg/l) 269 12 (5–27) Haemoglobin (g/dl) 269 12·7 (11·6–13·5) VAS (mm) 266 50 (30–67) DAS 28-ESR 265 5·0 (3·8–5·9) HAQ 243 1·5 (0·9–2·0) PLT (×103/mm3) 268 308 (251–379) Creatinine (mg/dl) 269 0·7 (0·6–0·8) N* n**(%) Sex (female) 272 254 (93%) RF presence 271 171 (63%) Anti-CCP presence 270 210 (78%) Morning stiffness 243 216 (89%) Organ symptoms 270 75 (28%) Coronary artery disease 266 28 (11%) Hypertension 268 110 (41%) Myocarditis 265 7 (3%) Diabetes 266 14 (5%) Renal failure 266 12 (5%) . RA patients . Characteristics . N* . Median (IQR) . Age (years) 268 56 (50–64) Disease duration (years) 265 10 (6–17) Larsen–Dale index 271 3 (3–4) Number of swollen joints 266 3 (1–7) Number of tender joints 266 8 (4–13) ESR (mm/h) 270 28 (15–42) CRP (mg/l) 269 12 (5–27) Haemoglobin (g/dl) 269 12·7 (11·6–13·5) VAS (mm) 266 50 (30–67) DAS 28-ESR 265 5·0 (3·8–5·9) HAQ 243 1·5 (0·9–2·0) PLT (×103/mm3) 268 308 (251–379) Creatinine (mg/dl) 269 0·7 (0·6–0·8) N* n**(%) Sex (female) 272 254 (93%) RF presence 271 171 (63%) Anti-CCP presence 270 210 (78%) Morning stiffness 243 216 (89%) Organ symptoms 270 75 (28%) Coronary artery disease 266 28 (11%) Hypertension 268 110 (41%) Myocarditis 265 7 (3%) Diabetes 266 14 (5%) Renal failure 266 12 (5%) Anti-CCP = anti-cyclic citrullinated peptide (CCP) antibodies; CRP = C-reactive protein; DAS-28 = disease activity score for 28 joints; ESR = erythrocyte sedimentation ratio; HAQ = Health Assessment Questionnaires (range 0–3); N* = number of patients with clinical information; n** = number of patients with positive clinical manifestation; PLT = platelet; RF = rheumatoid factor; VAS = visual analogue scale (range 0–100). Open in new tab Table 1 Baseline clinical and demographic features of the study patients with rheumatoid arthritis (RA). Data are expressed as median with interquartile ranges (IQR) or percentages . RA patients . Characteristics . N* . Median (IQR) . Age (years) 268 56 (50–64) Disease duration (years) 265 10 (6–17) Larsen–Dale index 271 3 (3–4) Number of swollen joints 266 3 (1–7) Number of tender joints 266 8 (4–13) ESR (mm/h) 270 28 (15–42) CRP (mg/l) 269 12 (5–27) Haemoglobin (g/dl) 269 12·7 (11·6–13·5) VAS (mm) 266 50 (30–67) DAS 28-ESR 265 5·0 (3·8–5·9) HAQ 243 1·5 (0·9–2·0) PLT (×103/mm3) 268 308 (251–379) Creatinine (mg/dl) 269 0·7 (0·6–0·8) N* n**(%) Sex (female) 272 254 (93%) RF presence 271 171 (63%) Anti-CCP presence 270 210 (78%) Morning stiffness 243 216 (89%) Organ symptoms 270 75 (28%) Coronary artery disease 266 28 (11%) Hypertension 268 110 (41%) Myocarditis 265 7 (3%) Diabetes 266 14 (5%) Renal failure 266 12 (5%) . RA patients . Characteristics . N* . Median (IQR) . Age (years) 268 56 (50–64) Disease duration (years) 265 10 (6–17) Larsen–Dale index 271 3 (3–4) Number of swollen joints 266 3 (1–7) Number of tender joints 266 8 (4–13) ESR (mm/h) 270 28 (15–42) CRP (mg/l) 269 12 (5–27) Haemoglobin (g/dl) 269 12·7 (11·6–13·5) VAS (mm) 266 50 (30–67) DAS 28-ESR 265 5·0 (3·8–5·9) HAQ 243 1·5 (0·9–2·0) PLT (×103/mm3) 268 308 (251–379) Creatinine (mg/dl) 269 0·7 (0·6–0·8) N* n**(%) Sex (female) 272 254 (93%) RF presence 271 171 (63%) Anti-CCP presence 270 210 (78%) Morning stiffness 243 216 (89%) Organ symptoms 270 75 (28%) Coronary artery disease 266 28 (11%) Hypertension 268 110 (41%) Myocarditis 265 7 (3%) Diabetes 266 14 (5%) Renal failure 266 12 (5%) Anti-CCP = anti-cyclic citrullinated peptide (CCP) antibodies; CRP = C-reactive protein; DAS-28 = disease activity score for 28 joints; ESR = erythrocyte sedimentation ratio; HAQ = Health Assessment Questionnaires (range 0–3); N* = number of patients with clinical information; n** = number of patients with positive clinical manifestation; PLT = platelet; RF = rheumatoid factor; VAS = visual analogue scale (range 0–100). Open in new tab The control group consisted of healthy volunteers who showed no clinical or laboratory signs of autoimmune disease. They were selected randomly from blood bank donors in order to match the patients in age, gender and ethnicity. Patients and control subjects had the same socio-economic status and were from the same geographical area. We selected a representative sample of the admixed urban Polish population. The study was approved by the Research Ethics Committee of the Institute of Rheumatology in Warsaw. DNA extraction Genomic DNA was extracted from whole blood collected in ethylenediamine tetraacetic acid (EDTA) tubes from patients with RA and the control group using the standard isothiocyanate guanidine (GTC) extraction method and/or the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Genotyping The TaqMan SNP genotyping assay (Applied Biosystems, Foster City, CA, USA) was used for genotyping of the NFATc2 (NC_000020·11) : rs880324 G/A polymorphism (assay ID: C_7596774_30), according to the manufacturer's instructions. Furthermore, SNPs in the Smad3 (NC_000015·10) gene, rs6494629 C/T and rs2289263 T/G, were determined using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. Amplification reaction was performed with 200 ng of genomic DNA in a 50-μl PCR mixture using 10 pmol of each primer, 0·25 mM each deoxyribonucleoside triphosphate (dNTP) (Qiagen), 1 U HotStar Taq polymerase (Qiagen) and ×1 PCR buffer (containing 1·5 μM magnesium chloride; Qiagen). Smad3 rs6494629 C/T The primer sequences were as follows: forward (5′-CAT CTT TCC TCC TGG CCA TA-3′) and reverse (5′-CTT AGC GAA GGA AAC CAG CA-3′). The parameters for PCR included an initial denaturing at 95°C for 15 min, followed by 35 cycles at 94°C for 30 s, 55·6°C for 60 s and 72°C for 60 s, with a final extension at 72°C for 10 min. Ten μl of PCR product [430 base pairs (bp) in length] was digested with 1 μl HpaII (Fermentas/Fisher Scientific, Loughborough, UK) at 37°C for 15 min, separated on a 2·5% agarose gel and visualized with ethidium bromide staining under ultraviolet light. HpaII digestion of the PCR product yielded 155 and 275 bp for allele C and 399 bp for the undigested allele T. Smad3 rs2289263 T/G The primer sequences were as follows: forward: 5′-TGC CTC TTC AGG GTC AGA CT-3′; reverse: 5′-CAA TGG AGG GGA ACG TTA AA-3′. The protocol for the PCR was as follows: 95°C for 15 min and 35 cycles of denaturing at 95°C for 30 s, annealing at 52°C for 60 s and extension at 72°C for 60 s, with a single final extension at 72°C for 10 min. Ten μl of PCR product (397 bp) was digested with 1 μl HphI (Fermentas) at 37°C for 360 min and separated on a 3% agarose gel and visualized with ethidium bromide staining under ultraviolet light. HphI digestion of PCR products yielded 142 and 237 bp for the T allele, whereas for allele G, 86, 142 and 151 bp fragments were observed. To confirm the accuracy of the method employed, randomly selected patients were analysed by direct sequencing, using an ABI PRISM Sequencer (Applied Biosystems). Detection of serum levels of Smad3 and NFATc2 For quantitative determination of Smad3/NFATc2 serum levels, samples from patients and controls were separated from peripheral venous blood and stored at −86°C until analysis. The levels of circulating of Smad3 and NFATc2 in serum were determined using commercially available enzyme-linked immunosorbent assay kits (ELISA; USCNK Life Science, Houston, TX, USA), according to the manufacturer's instructions. The minimum level of detection for Smad3 was 0·066 ng/ml and for NFATc2 was 5·9 pg/ml. Each sample was assayed in duplicate and the intra-assay coefficient of variation was <10%. The developed colour reaction was measured at optical density (OD)450 units on an ELISA reader (Elx800; Bio-Tek Instruments, Winooski, VT, USA). Statistical analysis Comparison of genotype distribution and allele frequencies between RA patients and the control group were estimated by computing odds ratios (ORs) and 95% confidence intervals (CIs). Statistical significance for the examined SNP comparison was set at P < 0·025 (according to Bonferroni correction). The association between target SNPs and the risk of RA was analysed by unconditional logistic regression using four genetic models, including co-dominant, dominant, recessive and over-dominant models evaluated using the χ2 test. For genetic association analyses, all polymorphisms were tested for deviations from the Hardy–Weinberg equilibrium (HWE) using the HardyWeinberg Simulator software (available at Institute of Human Genetics, Helmholtz Zentrum München, Germany). Linkage disequilibrium (LD), coefficient (D′ and r2) for haplotypes and their frequencies were performed using the genetic statistical software SHEsis, which is available at http://analysis2.bio-x.cn/myAnalysis.php. The association between SNPs and clinical/serological parameters was assessed by χ2 test with Yates' correction (categorical variables) or Mann–Whitney U-test (continuous variables). We used Bonferroni correction to adjust P-values for multiple measures. A Bonferroni-corrected α-level of P < 0·003 (0·05/16) was considered statistically significant. Results Polymorphisms in Smad3 and NFATc2 genes and risk of rheumatoid arthritis The distribution of genotype and allele frequencies of the polymorphisms rs6494629 C/T and rs2289263 T/G in Smad3 and rs880324 G/A in NFATc2 among patients and controls are shown in Table 2. The genotypes in RA patients for Smad3 rs6494629 and NFATC rs880324 and in controls for all examined SNPs were in HWE, whereas a significant deviation in genotype frequencies was observed in the RA patients for one of the SNPs – Smad3 rs2289263 (P < 0 0028). Genotyping (PCR–RFLP and sequencing) was repeated on randomly selected samples, giving complete conformity of the results. Table 2 Distribution of Smad3 and nuclear factor of activated T cells (NFAT)c2 alleles and genotypes in rheumatoid arthritis (RA) patients and healthy controls Smad3 rs6494629 (C/T) model . . RA, n (%) . Controls, n (%)* . OR (95% CI) . P-value . . Genotype . . . . . Co-dominant CC 54 (20%) 58 (18%) 1 – CT 127 (47%) 185 (58%) 0·74 (0·48–1·14) 0·168 TT 91 (33%) 78 (24%) 1·25 (0·78–2·02) 0·355 Dominant CC 54 (20%) 58 (18%) 1 CT+TT 218 (80%) 263 (82%) 0·89 (0·59–1·34) 0·580 Recessive CC+CT 181 (67%) 243 (76%) 1 TT 91 (33%) 78 (24%) 1·57 (1·09–2·24) 0·014 Over-dominant CC+TT 145 (53%) 136 (42%) 1 CT 127 (47%) 185 (58%) 0·64 (0·47–0·89) 0·008 Alleles C 235 (43%) 301 (47%) 1 T 309 (57%) 341 (53%) 1·16 (0·92–1·46) 0·204 Smad3 rs2289263 (T/G) model RA, n (%) Controls, n (%)** OR (95% CI) P-value Genotype Co-dominant TT 153 (56%) 175 (55%) 1 – TG 74 (27%) 112 (35%) 0·76 (0·53–1·09) 0·132 GG 45 (17%) 30 (10%) 1·72 (1·03–2·86) 0·037 Dominant TT 153 (56%) 175 (55%) 1 – TG+GG 119 (44%) 142 (54%) 0·96 (0·69–1·33) 0·799 Recessive TT+TG 227 (83%) 287 (90%) 1 – GG 45 (17%) 30 (10%) 1·90 (1·16–3·11) 0·010 Over-dominant TT+GG 198 (73%) 205 (65%) 1 – TG 74 (27%) 112 (35%) 0·68 (0·48–0·97) 0·034 Alleles T 380 (70%) 462 (73%) 1 – G 164 (30%) 172 (27%) 1·16 (0·90–1·49) 0·254 NFATc2 rs880324 (G/A) model RA, n (%) Controls, n (%)*** OR (95% CI) P-value Genotype Co-dominant GG 174 (64%) 198 (65%) 1 – GA 90 (33%) 95 (31%) 1·08 (0·76–1·53) 0·677 AA 8 (3%) 11 (4%) 0·83 (0·33–2·10) 0·691 Dominant GG 174 (64%) 198 (65%) 1 – GA+AA 98 (36%) 106 (35%) 1·05 (0·75–1·48) 0·771 Recessive GG+GA 264 (97%) 293 (96%) 1 AA 8 (3%) 11 (4%) 0·81 (0·32–2·04) 0·650 Over-dominant GG+AA 182 (67%) 209 (69%) 1 GA 90 (33%) 95 (31%) 1·09 (0·77–1·54) 0·637 Alleles G 438 (81%) 491 (81%) 1 A 106 (19%) 117 (19%) 1·02 (0·76–1·36) 0·917 Smad3 rs6494629 (C/T) model . . RA, n (%) . Controls, n (%)* . OR (95% CI) . P-value . . Genotype . . . . . Co-dominant CC 54 (20%) 58 (18%) 1 – CT 127 (47%) 185 (58%) 0·74 (0·48–1·14) 0·168 TT 91 (33%) 78 (24%) 1·25 (0·78–2·02) 0·355 Dominant CC 54 (20%) 58 (18%) 1 CT+TT 218 (80%) 263 (82%) 0·89 (0·59–1·34) 0·580 Recessive CC+CT 181 (67%) 243 (76%) 1 TT 91 (33%) 78 (24%) 1·57 (1·09–2·24) 0·014 Over-dominant CC+TT 145 (53%) 136 (42%) 1 CT 127 (47%) 185 (58%) 0·64 (0·47–0·89) 0·008 Alleles C 235 (43%) 301 (47%) 1 T 309 (57%) 341 (53%) 1·16 (0·92–1·46) 0·204 Smad3 rs2289263 (T/G) model RA, n (%) Controls, n (%)** OR (95% CI) P-value Genotype Co-dominant TT 153 (56%) 175 (55%) 1 – TG 74 (27%) 112 (35%) 0·76 (0·53–1·09) 0·132 GG 45 (17%) 30 (10%) 1·72 (1·03–2·86) 0·037 Dominant TT 153 (56%) 175 (55%) 1 – TG+GG 119 (44%) 142 (54%) 0·96 (0·69–1·33) 0·799 Recessive TT+TG 227 (83%) 287 (90%) 1 – GG 45 (17%) 30 (10%) 1·90 (1·16–3·11) 0·010 Over-dominant TT+GG 198 (73%) 205 (65%) 1 – TG 74 (27%) 112 (35%) 0·68 (0·48–0·97) 0·034 Alleles T 380 (70%) 462 (73%) 1 – G 164 (30%) 172 (27%) 1·16 (0·90–1·49) 0·254 NFATc2 rs880324 (G/A) model RA, n (%) Controls, n (%)*** OR (95% CI) P-value Genotype Co-dominant GG 174 (64%) 198 (65%) 1 – GA 90 (33%) 95 (31%) 1·08 (0·76–1·53) 0·677 AA 8 (3%) 11 (4%) 0·83 (0·33–2·10) 0·691 Dominant GG 174 (64%) 198 (65%) 1 – GA+AA 98 (36%) 106 (35%) 1·05 (0·75–1·48) 0·771 Recessive GG+GA 264 (97%) 293 (96%) 1 AA 8 (3%) 11 (4%) 0·81 (0·32–2·04) 0·650 Over-dominant GG+AA 182 (67%) 209 (69%) 1 GA 90 (33%) 95 (31%) 1·09 (0·77–1·54) 0·637 Alleles G 438 (81%) 491 (81%) 1 A 106 (19%) 117 (19%) 1·02 (0·76–1·36) 0·917 * Number of controls for Smad3 rs6494629 (C/T) = 321; ** number of controls for Smad3 rs2289263 (T/G) = 317; *** number of controls for NFATc2 rs880324 (G/A) = 304. CI = confidence interval; OR = odds ratio. Open in new tab Table 2 Distribution of Smad3 and nuclear factor of activated T cells (NFAT)c2 alleles and genotypes in rheumatoid arthritis (RA) patients and healthy controls Smad3 rs6494629 (C/T) model . . RA, n (%) . Controls, n (%)* . OR (95% CI) . P-value . . Genotype . . . . . Co-dominant CC 54 (20%) 58 (18%) 1 – CT 127 (47%) 185 (58%) 0·74 (0·48–1·14) 0·168 TT 91 (33%) 78 (24%) 1·25 (0·78–2·02) 0·355 Dominant CC 54 (20%) 58 (18%) 1 CT+TT 218 (80%) 263 (82%) 0·89 (0·59–1·34) 0·580 Recessive CC+CT 181 (67%) 243 (76%) 1 TT 91 (33%) 78 (24%) 1·57 (1·09–2·24) 0·014 Over-dominant CC+TT 145 (53%) 136 (42%) 1 CT 127 (47%) 185 (58%) 0·64 (0·47–0·89) 0·008 Alleles C 235 (43%) 301 (47%) 1 T 309 (57%) 341 (53%) 1·16 (0·92–1·46) 0·204 Smad3 rs2289263 (T/G) model RA, n (%) Controls, n (%)** OR (95% CI) P-value Genotype Co-dominant TT 153 (56%) 175 (55%) 1 – TG 74 (27%) 112 (35%) 0·76 (0·53–1·09) 0·132 GG 45 (17%) 30 (10%) 1·72 (1·03–2·86) 0·037 Dominant TT 153 (56%) 175 (55%) 1 – TG+GG 119 (44%) 142 (54%) 0·96 (0·69–1·33) 0·799 Recessive TT+TG 227 (83%) 287 (90%) 1 – GG 45 (17%) 30 (10%) 1·90 (1·16–3·11) 0·010 Over-dominant TT+GG 198 (73%) 205 (65%) 1 – TG 74 (27%) 112 (35%) 0·68 (0·48–0·97) 0·034 Alleles T 380 (70%) 462 (73%) 1 – G 164 (30%) 172 (27%) 1·16 (0·90–1·49) 0·254 NFATc2 rs880324 (G/A) model RA, n (%) Controls, n (%)*** OR (95% CI) P-value Genotype Co-dominant GG 174 (64%) 198 (65%) 1 – GA 90 (33%) 95 (31%) 1·08 (0·76–1·53) 0·677 AA 8 (3%) 11 (4%) 0·83 (0·33–2·10) 0·691 Dominant GG 174 (64%) 198 (65%) 1 – GA+AA 98 (36%) 106 (35%) 1·05 (0·75–1·48) 0·771 Recessive GG+GA 264 (97%) 293 (96%) 1 AA 8 (3%) 11 (4%) 0·81 (0·32–2·04) 0·650 Over-dominant GG+AA 182 (67%) 209 (69%) 1 GA 90 (33%) 95 (31%) 1·09 (0·77–1·54) 0·637 Alleles G 438 (81%) 491 (81%) 1 A 106 (19%) 117 (19%) 1·02 (0·76–1·36) 0·917 Smad3 rs6494629 (C/T) model . . RA, n (%) . Controls, n (%)* . OR (95% CI) . P-value . . Genotype . . . . . Co-dominant CC 54 (20%) 58 (18%) 1 – CT 127 (47%) 185 (58%) 0·74 (0·48–1·14) 0·168 TT 91 (33%) 78 (24%) 1·25 (0·78–2·02) 0·355 Dominant CC 54 (20%) 58 (18%) 1 CT+TT 218 (80%) 263 (82%) 0·89 (0·59–1·34) 0·580 Recessive CC+CT 181 (67%) 243 (76%) 1 TT 91 (33%) 78 (24%) 1·57 (1·09–2·24) 0·014 Over-dominant CC+TT 145 (53%) 136 (42%) 1 CT 127 (47%) 185 (58%) 0·64 (0·47–0·89) 0·008 Alleles C 235 (43%) 301 (47%) 1 T 309 (57%) 341 (53%) 1·16 (0·92–1·46) 0·204 Smad3 rs2289263 (T/G) model RA, n (%) Controls, n (%)** OR (95% CI) P-value Genotype Co-dominant TT 153 (56%) 175 (55%) 1 – TG 74 (27%) 112 (35%) 0·76 (0·53–1·09) 0·132 GG 45 (17%) 30 (10%) 1·72 (1·03–2·86) 0·037 Dominant TT 153 (56%) 175 (55%) 1 – TG+GG 119 (44%) 142 (54%) 0·96 (0·69–1·33) 0·799 Recessive TT+TG 227 (83%) 287 (90%) 1 – GG 45 (17%) 30 (10%) 1·90 (1·16–3·11) 0·010 Over-dominant TT+GG 198 (73%) 205 (65%) 1 – TG 74 (27%) 112 (35%) 0·68 (0·48–0·97) 0·034 Alleles T 380 (70%) 462 (73%) 1 – G 164 (30%) 172 (27%) 1·16 (0·90–1·49) 0·254 NFATc2 rs880324 (G/A) model RA, n (%) Controls, n (%)*** OR (95% CI) P-value Genotype Co-dominant GG 174 (64%) 198 (65%) 1 – GA 90 (33%) 95 (31%) 1·08 (0·76–1·53) 0·677 AA 8 (3%) 11 (4%) 0·83 (0·33–2·10) 0·691 Dominant GG 174 (64%) 198 (65%) 1 – GA+AA 98 (36%) 106 (35%) 1·05 (0·75–1·48) 0·771 Recessive GG+GA 264 (97%) 293 (96%) 1 AA 8 (3%) 11 (4%) 0·81 (0·32–2·04) 0·650 Over-dominant GG+AA 182 (67%) 209 (69%) 1 GA 90 (33%) 95 (31%) 1·09 (0·77–1·54) 0·637 Alleles G 438 (81%) 491 (81%) 1 A 106 (19%) 117 (19%) 1·02 (0·76–1·36) 0·917 * Number of controls for Smad3 rs6494629 (C/T) = 321; ** number of controls for Smad3 rs2289263 (T/G) = 317; *** number of controls for NFATc2 rs880324 (G/A) = 304. CI = confidence interval; OR = odds ratio. Open in new tab Four genetic models, co-dominant, dominant, recessive and over-dominant, were applied to assess the association of SNPs within the Smad3 and NFATc2 gene and RA risk. With regard to the rs6494629 C/T Smad3 gene polymorphism, RA patients showed significantly different genotype and allele distributions compared to control subjects in two models. In the recessive model, the frequency of the TT genotype was significantly higher and that of the CC+CT genotypes was lower in RA patients compared to the healthy subjects (OR = 1·57, CI = 1·09–2·24; P = 0·014). In the over-dominant model (CC+TT versus CT), the association was also significant (OR = 0·64, CI = 0·47–0·89; P = 0·008). No statistically significant differences were observed when allele distribution was compared between RA patients and healthy controls (OR = 1·16, CI = 0·92–1·46; P = 0·204). The analysis of the Smad3 rs2289263 T/G polymorphism revealed significant differences in the case–control distribution in various models. In the co-dominant model, the frequency of the GG genotype was significantly higher (OR = 1·72, CI = 1·03–2·86; P = 0·037) in RA patients compared to the healthy subjects. In the recessive model, the frequency of the GG genotype was higher and that of the TT+TG genotypes was lower in RA patients compared to the healthy subjects (OR = 1·90, CI = 1·16–3·1; P = 0·010) Similarly, in the over-dominant model (TT+GG versus TG), the association was also significant (OR = 0·68, CI = 0·48–0·97; P = 0·034). However, no statistically significant differences were observed in the allele distribution between RA patients and healthy controls. We observed no significant differences in genotype and allele frequencies of the NFATc2 rs880324 G/A variants between RA patients and controls in all models examined (Table 2). Haplotype analysis of the Smad3 polymorphisms The haplotypes were also explored to determine whether any particular haplotype may be associated with risk of RA. In this report, Smad3 haplotypes were assessed for the RA group and the control group. Analysis by the SHEsis program demonstrated that rs6494629 C/T and rs2289263 T/G Smad3 gene polymorphisms were in a weak LD with D′ = 0·116 and r2 = 0·004. When both SNPs were assessed in haplotype analysis, four potential haplotypes were formed (Table 3). There was a predominance of the TG haplotype in the cases compared to controls. The TG haplotype indicated a significantly higher risk for rheumatoid arthritis (OR = 1·39, CI = 1·01–1·91; P = 0·041). In contrast, the frequencies of other haplotypes, CG, CT and TT, were decreased in the RA patients compared with healthy subjects. However, these differences were not significant. Table 3 Smad3 haplotypes in rheumatoid arthritis (RA) patients and controls Haplotype . RA 2n = 556 (%) . Controls 2n = 532(%) . P . OR (95% CI) . CG 61·48 (11·1) 64·04 (12·0) 0·612 0·91 (0·63–1·32) CT 178·52 (32·1) 184·96 (34·8) 0·352 0·89 (0·69–1·14) TG 108·52 (19·5) 78·96 (14·8) 0·041 1·39 (1·01–1·91) TT 207·48 (37·3) 204·04 (38·4) 0·724 0·96 (0·75–1·22) Haplotype . RA 2n = 556 (%) . Controls 2n = 532(%) . P . OR (95% CI) . CG 61·48 (11·1) 64·04 (12·0) 0·612 0·91 (0·63–1·32) CT 178·52 (32·1) 184·96 (34·8) 0·352 0·89 (0·69–1·14) TG 108·52 (19·5) 78·96 (14·8) 0·041 1·39 (1·01–1·91) TT 207·48 (37·3) 204·04 (38·4) 0·724 0·96 (0·75–1·22) P = Fisher's test; P considered significant shown in italic type. CI = confidence interval; OR = odds ratio. Open in new tab Table 3 Smad3 haplotypes in rheumatoid arthritis (RA) patients and controls Haplotype . RA 2n = 556 (%) . Controls 2n = 532(%) . P . OR (95% CI) . CG 61·48 (11·1) 64·04 (12·0) 0·612 0·91 (0·63–1·32) CT 178·52 (32·1) 184·96 (34·8) 0·352 0·89 (0·69–1·14) TG 108·52 (19·5) 78·96 (14·8) 0·041 1·39 (1·01–1·91) TT 207·48 (37·3) 204·04 (38·4) 0·724 0·96 (0·75–1·22) Haplotype . RA 2n = 556 (%) . Controls 2n = 532(%) . P . OR (95% CI) . CG 61·48 (11·1) 64·04 (12·0) 0·612 0·91 (0·63–1·32) CT 178·52 (32·1) 184·96 (34·8) 0·352 0·89 (0·69–1·14) TG 108·52 (19·5) 78·96 (14·8) 0·041 1·39 (1·01–1·91) TT 207·48 (37·3) 204·04 (38·4) 0·724 0·96 (0·75–1·22) P = Fisher's test; P considered significant shown in italic type. CI = confidence interval; OR = odds ratio. Open in new tab Association between the Smad3 rs6494629 C/T and rs2289263 T/G polymorphisms and RA phenotype To explore possible relationships between the Smad3 gene SNPs and clinical/biochemical parameters in RA patients, we performed a stratified analysis of combined genotypes with the rs6494629 CC genotype versus the CT+TT genotype and rs2289263 TT genotype versus the TG+GG genotype. Without Bonferroni correction, the genotype–phenotype analysis showed correlation of the Smad3 rs6494629 C/T polymorphism with age, mean value of platelet (PLT), Health Assessment Questionnaire (HAQ) score and rheumatoid factor (RF) presence (Supporting information, Table S1). All these parameters were significantly higher in RA patients with the combined genotype rs6494629 CT+TT in comparison to patients with the wild-type genotype (P = 0·017, 0·045, 0·014 and 0·026; respectively). However, after Bonferroni correction for multiple testing there was no significant association between Smad3 rs6494629 C/T SNP and RA phenotype (Table 4 and Supporting information, Table S2). In our study we also performed multivariate logistic regression analysis for some clinical parameters to identify factors associated with the Smad3 rs6494629 CC polymorphism in RA patients. Joint counts, ESR and CRP were not included into the multivariate model because they are components of the disease activity score for 28 joints (DAS28). Data are shown in Supporting information, Table S3. We observed an association of PLT and RF with the rs6494629 CC genotype. The mean value of PLT and RF presence was lower in RA patients with the Smad3 rs6494629 CC genotype in comparison to patients with the Smad3 rs6494629 T allele (Table 4). Table 4 The disease activity and laboratory parameters in relation to Smad3 rs6494629 (C/T) in rheumatoid arthritis (RA) patients; CC versus CT+TT, with Bonferroni correction . CC . CT + TT . . Parameter . n . Median (IQR) . n . Median (IQR) . P* . Age (years) 53 53 (41–62) 215 57 (51–65) 0·017 Disease duration (years) 51 12 (6–20) 214 10 (6–17) 0·832 Larsen–Dale index 54 3 (2–4) 217 3 (3–4) 0·855 ESR (mm/h) 54 24·5 (14–36) 216 29 (15–43·5) 0·182 Number of swollen joints 52 2 (0–7) 214 3 (1–7) 0·282 Number of tender joints 52 8 (2–13) 214 8 (4–13) 0·496 CRP (mg/l) 54 11·4 (5–30·4) 215 12 (5–25·8) 0·455 Haemoglobin (g/dl) 54 12·7 (11·9–13·5) 215 12·7 (11·5–13·5) 0·942 VAS (mm) 52 48 (24–65) 214 51 (31–69) 0·170 DAS-28 52 4·3 (3·4–5·8) 213 5·0 (4·0–5·9) 0·125 PLT (×103/mm3) 53 288 (240–340) 215 316 (259–386) 0·045 Creatinine (mg/dl) 53 0·7 (0·6–0·7) 216 0·7 (0·6–0·8) 0·348 HAQ 47 1·0 (0·63–1·75) 196 1·5 (1·0–2·0) 0·014 CC CT + TT N n (%) N n (%) P** Women 54 51 (94%) 218 203 (93%) 0·964 RF presence 54 27 (50%) 217 144 (66%) 0·026 Anti-CCP presence 53 40 (76%) 217 170 (78%) 0·652 . CC . CT + TT . . Parameter . n . Median (IQR) . n . Median (IQR) . P* . Age (years) 53 53 (41–62) 215 57 (51–65) 0·017 Disease duration (years) 51 12 (6–20) 214 10 (6–17) 0·832 Larsen–Dale index 54 3 (2–4) 217 3 (3–4) 0·855 ESR (mm/h) 54 24·5 (14–36) 216 29 (15–43·5) 0·182 Number of swollen joints 52 2 (0–7) 214 3 (1–7) 0·282 Number of tender joints 52 8 (2–13) 214 8 (4–13) 0·496 CRP (mg/l) 54 11·4 (5–30·4) 215 12 (5–25·8) 0·455 Haemoglobin (g/dl) 54 12·7 (11·9–13·5) 215 12·7 (11·5–13·5) 0·942 VAS (mm) 52 48 (24–65) 214 51 (31–69) 0·170 DAS-28 52 4·3 (3·4–5·8) 213 5·0 (4·0–5·9) 0·125 PLT (×103/mm3) 53 288 (240–340) 215 316 (259–386) 0·045 Creatinine (mg/dl) 53 0·7 (0·6–0·7) 216 0·7 (0·6–0·8) 0·348 HAQ 47 1·0 (0·63–1·75) 196 1·5 (1·0–2·0) 0·014 CC CT + TT N n (%) N n (%) P** Women 54 51 (94%) 218 203 (93%) 0·964 RF presence 54 27 (50%) 217 144 (66%) 0·026 Anti-CCP presence 53 40 (76%) 217 170 (78%) 0·652 P* = Mann–Whitney U-test; P** = χ2 test. P < 0·003 (Bonferroni correction) was considered significant. Anti-CCP = anti-cyclic citrullinated peptide; n = number of patients with clinical information; IQR = interquartile range; CRP = C-reactive protein; DAS-28 = disease activity score for 28 joints; ESR = erythrocyte sedimentation ratio; HAQ = Health Assessment Questionnaires; PLT = platelet; RF = rheumatoid factor; VAS = visual analogue scale. Open in new tab Table 4 The disease activity and laboratory parameters in relation to Smad3 rs6494629 (C/T) in rheumatoid arthritis (RA) patients; CC versus CT+TT, with Bonferroni correction . CC . CT + TT . . Parameter . n . Median (IQR) . n . Median (IQR) . P* . Age (years) 53 53 (41–62) 215 57 (51–65) 0·017 Disease duration (years) 51 12 (6–20) 214 10 (6–17) 0·832 Larsen–Dale index 54 3 (2–4) 217 3 (3–4) 0·855 ESR (mm/h) 54 24·5 (14–36) 216 29 (15–43·5) 0·182 Number of swollen joints 52 2 (0–7) 214 3 (1–7) 0·282 Number of tender joints 52 8 (2–13) 214 8 (4–13) 0·496 CRP (mg/l) 54 11·4 (5–30·4) 215 12 (5–25·8) 0·455 Haemoglobin (g/dl) 54 12·7 (11·9–13·5) 215 12·7 (11·5–13·5) 0·942 VAS (mm) 52 48 (24–65) 214 51 (31–69) 0·170 DAS-28 52 4·3 (3·4–5·8) 213 5·0 (4·0–5·9) 0·125 PLT (×103/mm3) 53 288 (240–340) 215 316 (259–386) 0·045 Creatinine (mg/dl) 53 0·7 (0·6–0·7) 216 0·7 (0·6–0·8) 0·348 HAQ 47 1·0 (0·63–1·75) 196 1·5 (1·0–2·0) 0·014 CC CT + TT N n (%) N n (%) P** Women 54 51 (94%) 218 203 (93%) 0·964 RF presence 54 27 (50%) 217 144 (66%) 0·026 Anti-CCP presence 53 40 (76%) 217 170 (78%) 0·652 . CC . CT + TT . . Parameter . n . Median (IQR) . n . Median (IQR) . P* . Age (years) 53 53 (41–62) 215 57 (51–65) 0·017 Disease duration (years) 51 12 (6–20) 214 10 (6–17) 0·832 Larsen–Dale index 54 3 (2–4) 217 3 (3–4) 0·855 ESR (mm/h) 54 24·5 (14–36) 216 29 (15–43·5) 0·182 Number of swollen joints 52 2 (0–7) 214 3 (1–7) 0·282 Number of tender joints 52 8 (2–13) 214 8 (4–13) 0·496 CRP (mg/l) 54 11·4 (5–30·4) 215 12 (5–25·8) 0·455 Haemoglobin (g/dl) 54 12·7 (11·9–13·5) 215 12·7 (11·5–13·5) 0·942 VAS (mm) 52 48 (24–65) 214 51 (31–69) 0·170 DAS-28 52 4·3 (3·4–5·8) 213 5·0 (4·0–5·9) 0·125 PLT (×103/mm3) 53 288 (240–340) 215 316 (259–386) 0·045 Creatinine (mg/dl) 53 0·7 (0·6–0·7) 216 0·7 (0·6–0·8) 0·348 HAQ 47 1·0 (0·63–1·75) 196 1·5 (1·0–2·0) 0·014 CC CT + TT N n (%) N n (%) P** Women 54 51 (94%) 218 203 (93%) 0·964 RF presence 54 27 (50%) 217 144 (66%) 0·026 Anti-CCP presence 53 40 (76%) 217 170 (78%) 0·652 P* = Mann–Whitney U-test; P** = χ2 test. P < 0·003 (Bonferroni correction) was considered significant. Anti-CCP = anti-cyclic citrullinated peptide; n = number of patients with clinical information; IQR = interquartile range; CRP = C-reactive protein; DAS-28 = disease activity score for 28 joints; ESR = erythrocyte sedimentation ratio; HAQ = Health Assessment Questionnaires; PLT = platelet; RF = rheumatoid factor; VAS = visual analogue scale. Open in new tab Moreover, we observed that the carriers of at least one polymorphic allelic variant of the Smad3 rs6494629 (CT or TT genotypes) had higher parameters of disease activity and joint damage than patients with the wild-type genotype. Analysis of the second Smad3 gene polymorphism at position rs2289263 T/G, without Bonferroni correction, showed that the duration of RA was significantly higher in carriers of the combined rs2289263 TG+GG genotype in comparison with rs2289263 TT subjects (P = 0 0019; data not shown). However, after Bonferroni correction for multiple testing there was no significant association between Smad3 rs2289263 T/G polymorphism and RA phenotype (data not shown). Also, no association could be detected between the Smad3 rs6494629 C/T and rs2289263 T/G variants and other disease activity and laboratory parameters and extra-articular manifestation (ExRA) among RA patients. Association of the NFATc2 rs880324 G/A gene polymorphisms with clinical manifestation of RA We found no significant differences in genotype distribution of the rs880324 G/A NFATc2 gene polymorphism among RA patients divided according to the disease activity, joint damage, laboratory parameters and extra-articular manifestation (data not shown). Smad3 and NFATc2 levels in patients/controls and in relation to RA clinical parameters Samples were stratified into positive and negative based on detected levels of Smad3 and NFATc2 (>0·066 ng/ml and >5·9 pg/ml, respectively). As shown in Table 5 (and Supporting information, Figs S1 and S2), the number of Smad3- and NFATc2-positive RA patients was significantly higher than the number of positive healthy subjects (P < 0·001 in both cases). We next conducted a comparative analysis between positive and negative patients in relation to clinical parameters and ExRA. However, correlation analysis did not show any significant relationship between the studied serum protein levels and clinical and biochemical parameters in our RA patients (data not shown). Table 5 Smad3 and nuclear factor of activated T cells (NFAT)c2 protein level in rheumatoid arthritis (RA) patients and healthy subjects Protein level . RA median (IQR) . Control median (IQR) . P . Smad3 (g/ml) 0·350 (0·214–0·557) 0·104 (0·029–0·224) <0·001 NFATc2 9pg/ml) 720 (468–972) 201 (73–346) <0·001 Protein level . RA median (IQR) . Control median (IQR) . P . Smad3 (g/ml) 0·350 (0·214–0·557) 0·104 (0·029–0·224) <0·001 NFATc2 9pg/ml) 720 (468–972) 201 (73–346) <0·001 P = Mann–Whitney U-test, RA versus controls. P ≤ 0 05 was considered significant. IQR = interquartile range. Open in new tab Table 5 Smad3 and nuclear factor of activated T cells (NFAT)c2 protein level in rheumatoid arthritis (RA) patients and healthy subjects Protein level . RA median (IQR) . Control median (IQR) . P . Smad3 (g/ml) 0·350 (0·214–0·557) 0·104 (0·029–0·224) <0·001 NFATc2 9pg/ml) 720 (468–972) 201 (73–346) <0·001 Protein level . RA median (IQR) . Control median (IQR) . P . Smad3 (g/ml) 0·350 (0·214–0·557) 0·104 (0·029–0·224) <0·001 NFATc2 9pg/ml) 720 (468–972) 201 (73–346) <0·001 P = Mann–Whitney U-test, RA versus controls. P ≤ 0 05 was considered significant. IQR = interquartile range. Open in new tab Serum Smad3 and NFATc2 levels in respect to examined polymorphisms To evaluate whether Smad3 and NFATc2 polymorphisms are associated with dysregulation of Smad3 and NFATc2, respectively, we conducted ELISA assays of sera of RA patients and healthy subjects at protein levels. First, we examined the relationship between Smad3/NFATc2 expression levels in RA patients and the control group in relation to rs6494629 C/T and rs2289263 T/G Smad3 and rs880324 G/A NFATc2 genotypes. In this case, we found no significant association, either among RA patients or in healthy subjects (data not shown). We next conducted a comparative analysis between RA patients and the control group according to rs6494629 C/T and rs2289263 T/G Smad3 and rs880324 G/A NFATc2 genotypes. Serum levels of Smad3 in RA patients with rs6494629 CC, CT and TT genotypes were significantly higher than healthy subjects with the same rs6494629C/T genotypes (all P < 0·001; Fig. 1a). Increased serum levels of Smad3 were also observed in RA patients with rs2289263 TT, TG and GG genotypes compared to controls (all P < 0·001, Fig. 1b). Moreover, serum levels of NFATc2 in RA patients with rs880324 GG, GA and AA genotypes were significantly higher than detected in the sera of healthy donors (P < 0·001, P < 0·001, P = 0·004; Fig. 1c). Fig. 1 Open in new tabDownload slide Variation in Smad3 and nuclear factor of activated T cells (NFAT)c2 expression levels in rheumatoid arthritis (RA) patients and control group in relation to: (a) rs6494629 C/T Smad3 genotypes. RA versus control group: TT genotype: P < 0·001, CT genotype: P < 0·001, CC genotype: P < 0·001; (b) rs2289263 T/G Smad3 genotypes. RA versus control group: TT genotype: P < 0·001, GT genotype: P < 0·001, GG genotype: P < 0·001; (c) rs880324 G/A NFATc2 genotypes. RA versus control group: GG genotype: P < 0·001, GA genotype: P < 0·001, AA genotype: P = 0·004. Fig. 1 Open in new tabDownload slide Variation in Smad3 and nuclear factor of activated T cells (NFAT)c2 expression levels in rheumatoid arthritis (RA) patients and control group in relation to: (a) rs6494629 C/T Smad3 genotypes. RA versus control group: TT genotype: P < 0·001, CT genotype: P < 0·001, CC genotype: P < 0·001; (b) rs2289263 T/G Smad3 genotypes. RA versus control group: TT genotype: P < 0·001, GT genotype: P < 0·001, GG genotype: P < 0·001; (c) rs880324 G/A NFATc2 genotypes. RA versus control group: GG genotype: P < 0·001, GA genotype: P < 0·001, AA genotype: P = 0·004. Discussion Before any molecular genetic studies were performed, a number of epidemiological investigations had provided compelling evidence for a genetic component to rheumatoid arthritis, placing it into the category of multi-factorial/polygenic disorders [21]. Although several genes have been pinpointed as susceptibility markers of the disease [22], there is no recognized hereditary profile that might confirm this association and cannot be performed with sufficient accuracy to enter clinical practice [23,24]. We now know that the disease is driven at least partially by CD4+ T cells, which specifically express many of the genes located within RA-associated loci [25]. Moreover, polymorphisms in the T cell-expressed genes, including transcriptional factors, are now recognized to confer increased risk not only for RA, but also other autoimmune diseases. Thus, Smad3 and NFATc2 play a crucial role in transcriptional activation/regulation in T lymphocytes; however, little is known about the role of Smad3 and NFATc2 gene polymorphisms in autoimmune diseases, including RA. The Smad3 polymorphisms have been associated with susceptibility to atopic dermatitis [26], osteoarthritis [27,28], Kawasaki disease [29] and inflammatory bowel diseases [30] and with cardiovascular events in RA patients [31], but not type 1 diabetes mellitus [32] and juvenile polyposis syndrome [33]. In the sole report exploring Smad3 gene polymorphisms in osteoarthritis (OA), Valdes et al. [28] studied the 10 selected Taq SNPs in the intronic region of Smad3 gene. They concluded that the SNPs rs12901499, rs6494629 and rs2289263 are involved in genetic susceptibility to large-joint OA in the UK population. The study by Garci-Bermudez et al. [31] in the Spanish RA population showed a potential protective effect of the C allele of the Smad3 rs17228212 polymorphism against the risk of developing cardiovascular events in anti-cyclic citrullinated peptide (CCP)-negative patients. Furthermore, variation in the NFATc2 gene has not been linked with any autoimmune phenotype; however, mice lacking NFATc2 show increased inflammation in experimentally induced allergic asthma [34,35] and over-expression of specific matrix-degrading proteinases and proinflammatory cytokines in experimentally induced osteoarthritis [15]. Considering differences in genetic predispositions between populations and an important role of the Smad3/NFATc2 in the functioning of the T helper type 17/regulatory T cells (Th17/Treg) cells [8,11], we decided to carry out an analysis of selected polymorphisms located in this gene in relation to RA. To our knowledge, this is the first report to determine whether the Smad3 (rs6494629 C/T and rs2289263 T/G) and NFATc2 (rs880324 G/A) gene polymorphisms and their protein levels were associated with RA. All selected genetic variants are located in an intronic region with no obvious function. Further studies are needed to identify the functional role of these variants and/or to determine if some of these SNPs are in LD with other variants which affect the level/function of the Smad3 gene product. A comparison of our data with the HapMap database (http://hapmap.ncbi.nlm.nih.gov/) revealed some differences in the distribution of Smad3 and NFATc2 gene polymorphisms among ethnic groups. In our controls Smad3 rs6494629 C/T allele frequencies were similar to those of the United States, Africa and other European populations, whereas rs2289263 T/G were distinct (lower frequency of polymorphic G allele in Polish healthy subjects than in other populations: 27 versus 32–49%). Furthermore, the NFATc2 minor rs880324 A allele frequency was lower in Polish subjects (19%) than in populations from Africa and Europe (from 22 to 32%) but, at the same time, higher than in other populations (0·5–14%). Discrepancies in allele/genotype frequencies between reports may be explained in part by the ethnic variability between populations, heterogeneity of the analysed diseases and genetic trait differences, as well as the limited sample size. The results of this study also showed a significant difference between RA patients and healthy subjects in genotype distribution and allele frequencies for both Smad3 gene-examined polymorphisms. The subjects with the rs6494629 TT and rs2289263 GG genotypes were at a higher risk for RA than those with the rs6494629 CC and rs2289263 TT genotypes, indicating that polymorphic alleles of both SNPs in Smad3 gene are putative RA risk alleles in the Polish population. Additionally, haplotype analysis has also shown that carriers of both polymorphic alleles rs6494629 T/rs2289263G had a significantly higher risk for RA compared with carriers of other haplotypes. Moreover, our detailed genotype–phenotype analysis indicated that the combined Smad3 rs6494629 C/T variant genotype (CT + TT) was associated with a significantly higher age, mean value of PLT count, HAQ score and RF presence without Bonferroni correction. When Bonferroni correction for multiple testing was used we found no significant association between SNP and RA phenotype in our patients. In addition, we observed that RA patients with the rare allelic variant Smad3 rs6494629 T had more advanced disease than wild-type allele carriers, suggesting that this SNP might be associated with increased disease activity. We hypothesize that the Smad3 gene polymorphisms may lead to disturbances in the TGF/Smad signalling pathway, which plays an important role in the regulation of T cell responses and gene expression via multiple mechanisms. This can lead to inhibition of the transforming growth factor (TGF)-β-mediated induction of forkhead box protein 3 (FoxP3)-expressing Treg cells, as well as induction of Th1 and Th17 development. Unfortunately, in our study we found no evidence for any association of the NFATc2 rs880324 G/A polymorphism with susceptibility to RA and its severity, although the relatively small sample size may have provided insufficient statistical power. To examine the effect of SNP analysis on Smad3 and NFATc2 gene expression, we determined the Smad3/NFATc2 expression levels in the serum of RA patients and healthy donors. We observed that both Smad3 and NFATc2 serum levels were significantly higher in RA patients than in healthy subjects, reflecting the ongoing inflammatory process in patients and the attempts to keep it under control. Several previous reports have described Smad3 or NFATc2 mRNA and/or protein levels [7,26,36–39], but our report is the first, to our knowledge, to explore the protein expression of both in the peripheral blood of RA patients. We observed that RA patients had significantly higher Smad3 and NFATc2 protein levels than controls, but we found no correlation between both protein levels and clinical parameters and examined SNPs. However, we observed that in Smad3-positive patients both polymorphic alleles rs6494629 T/rs2289263G occurred more frequently and they had higher parameters of disease activity and joint damage compared to Smad3-negative patients. Smad3 protein defects may cause the reduction of immunological responses, decrease bone mineral density, increase the expression of metalloproteinases and increase the expression of proinflammatory cytokines and chemokines by Th1 and Th17 cells [19,20]. The present study has some limitations. First, a larger sample size in different populations is required to validate the results. Secondly, information is lacking on the Smad3/NFATc2 protein and/or mRNA levels in T cells or in peripheral blood mononuclear cells (PMBCs), which could provide us with more information about the role of the Smad3/NFATc2 in the pathogenesis of RA. Thirdly, significant deviation in genotype frequencies was observed in the RA patients for one of the SNPs – Smad3 rs2289263 (P < 0 0028). Deviations from HWE can be extremely informative. It could imply a sampling bias, mistyping of genotypes, ethnic differences and migration as well as population stratification, and/or combinations of these reasons. Although deviation from the HWE often indicates poor genotyping quality, it can also be caused by a small sample size. Minimization of genotyping errors was achieved by repetition of testing and sequencing of randomly selected samples. Recent years have seen increasing efforts to understand the involvement of T cells in the immunopathogenesis of RA. This has led to a rising interest in the possibility of using Treg cells in biological therapy for controlling autoimmunity. Therefore, finding the genetic basis of autoimmune disorders may constitute a step towards understanding the immunology of the pathogenic process leading to the disease. Our study is the first protocol, which evaluated the possible association between Smad3/NFATc2 genotypes and RA. The results suggest that the Smad3 rs6494629 C/T and rs2289263T/G polymorphisms may be associated with susceptibility to RA. Further studies are necessary to identify the association of these variants with RA in our and other populations and to provide a global view of these SNPs in the pathogenesis of RA. Acknowledgements We are grateful to all the RA patients whose co-operation made this study possible. We are also grateful to Wieslawa Frankowska and Teresa Golaszewska for technical assay. This work was supported by a grant from the Polish National Science Center (2011/01/D/NZ5/01396). Disclosure All authors state that they have no conflicts of interest related to this work. References 1 Lin SC , Chen KH, Lin CH, Kuo CC, Ling QD, Chan CH. The quantitative analysis of peripheral blood FOXP3-expresing T cells in systemic lupus erythematous and rheumatoid arthritis patients . Eur J Clin Invest 2007 ; 37 : 987 – 996 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Akil M , Amos RS. ABC of rheumatology: rheumatoid arthritis – I: clinical features and diagnosis . BMJ 1995 ; 310 : 587 – 590 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Sangha O . Epidemiology of rheumatic diseases . Rheumatology 2000 ; 39 ( Suppl. 2 ): 3 – 12 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Silman A , Pearson JE. Epidemiology and genetics of rheumatoid arthritis . 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TI - Association of the Smad3 and NFATc2 gene polymorphisms and their serum levels with susceptibility to rheumatoid arthritis in Polish cohorts
JF - Clinical & Experimental Immunology
DO - 10.1111/cei.12482
DA - 2015-02-16
UR - https://www.deepdyve.com/lp/oxford-university-press/association-of-the-smad3-and-nfatc2-gene-polymorphisms-and-their-serum-Q2qFQMAstk
SP - 444
EP - 453
VL - 179
IS - 3
DP - DeepDyve
ER -