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Signal transducer and activator of transcription 4 gene polymorphisms associated with rheumatoid arthritis in Northwestern Chinese Han population
Life Sciences 89 (2011) 171–175
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Signal transducer and activator of transcription 4 gene polymorphisms associated with rheumatoid arthritis in Northwestern Chinese Han population Ya-ling Liang a, Hua Wu a, Pei-qiang Li a, Xiao-dong Xie a,⁎, Xi Shen a, Xiao-qing Yang b, Xuan Cheng c, Li Liang d a
Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou City, 730000, Gansu Province, China Department of internal medicine, the affiliated Lanzhou Petrochemical hospital, Lanzhou City, 730060, Gansu Province, China Department of Laboratory Medicine, Traditional Chinese Medicine hospital of Gansu Province, Lanzhou City, 730050, Gansu Province, China d Department of Pharmacy, the first hospital of Lanzhou University, Lanzhou City, 730000, Gansu Province, China b c
a r t i c l e
i n f o
Article history: Received 4 October 2010 Accepted 19 May 2011 Keywords: STAT4 Rheumatoid arthritis Single nucleotide polymorphisms Case–control study
a b s t r a c t Aims: Signal transducer and activator of transcription 4 (STAT4) gene encode a transcriptional factor that transmits signals induced by several key cytokines which play important roles in the development of autoimmune diseases. Recently, several single nucleotide polymorphisms (SNPs) in STAT4 gene have been reported to be significantly associated with Rheumatoid arthritis (RA) in different ethnic populations. We undertook this study to investigate whether the association of STAT4 genetic polymorphisms with RA is present in Northwestern Chinese Han population. Main methods: A case–control association study in individuals with RA (n = 208) and healthy controls (n = 312) was conducted. Four SNPs (rs7574865, rs8179673, rs10181656, rs11889341) in STAT4 gene were genotyped by using polymerase chain reaction followed by denaturing high-performance liquid chromatography (PCR-DHPLC) and DNA sequencing. Key findings: The genotype and allele distributions of four polymorphisms were significantly different in individuals with RA compared to controls, with SNP rs7574865 T allele and T/T genotype showing the most significant association with susceptibility to RA (uncorrected P = 1 × 10− 4, OR = 1.645, 95% CI = 1.272–2.129; uncorrected P = 4.8 × 10− 5, OR = 3.111, 95% CI = 1.777–5.447, respectively). Stratification studies showed that STAT4 gene polymorphisms were significantly associated with anti-cyclic citrullinated peptide (antiCCP) positive subgroup in Northwestern Chinese Han population. Significance: These findings strongly suggest that STAT4 genetic polymorphisms are associated with RA in Northwestern Chinese Han population, and support the hypothesis of STAT4 gene polymorphisms increasing the risk for RA across major populations. © 2011 Elsevier Inc. All rights reserved.
Introduction Rheumatoid arthritis (RA) is a common chronic inflammatory autoimmune disease characterized by significant disability and early mortality, which affects ~1% of the adult population worldwide (Silman and Pearson, 2002). It is generally accepted that RA is a complex disease, with suspected interrelated contributions from genetic, infectious, environmental and hormonal factors (Firestein, 2003). Twin and family studies suggest that genetic factors contribute up to 60% of disease susceptibility to RA (MacGregor et al., 2000). To date, human leukocyte antigen (HLA) class II molecules have been documented as the most powerful genetic factors of RA across all populations, but they account for no more than one-third of the total
⁎ Corresponding author. Tel.: + 86 931 8915023; fax: + 86 931 8915001. E-mail address: [email protected] (X. Xie). 0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.05.012
genetic susceptibility (Newton et al., 2004; Cornelis et al., 1998). Recently, several non-HLA genes have been identified to contribute to RA susceptibility (Bowes and Barton, 2008). However, subsequent studies in European and Asian populations have got conflicting results (Yamada and Yamamoto, 2007; Kyogoku et al., 2004; Ikari et al., 2006; Orozco et al., 2005), which suggest that the genetic backgrounds of different ethnic groups should be taken into account (Colhoun et al., 2003). Signal transducer and activator of transcription 4 (STAT4) gene encode a transcription factor which resides in cytosol and transmits the intracellular signals induced by cytokines including interleukin-12 (IL-12), IL-23, IL-27 and type 1 interferons (IFNs). Upon cytokine signaling, this transcription factor becomes phosphorylated and translocates to the nucleus to play an essential downstream role in the differentiation and proliferation of IL-12-dependent T helper 1 (Th1) cells (Watford et al., 2004). STAT4 is also important in the development of IL-17-secreting Th cells (Th17) in response to IL-23 (Mathur et al., 2007). Since Th1 and Th17 lineages work as crucial
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effectors in chronic inflammatory disorders, STAT4 gene may play an important role in the pathogenesis of RA (McInnes and Schett, 2007; Thierfelder et al., 1996). Currently, several single nucleotide polymorphisms (SNPs) in STAT4 gene have been reported to be significantly associated with RA and systemic lupus erythematosus (SLE) (Amos et al., 2008; Remmers et al., 2007). And subsequent association studies between STAT4 gene polymorphisms and RA in Caucasians and Asians have yielded consistent results (Lee et al., 2007; Palomino-Morales et al., 2008; Barton et al., 2008; Kobayashi et al., 2008; Orozco et al., 2008; Martinez et al., 2008; Daha et al., 2009; Suarez-Gestal et al., 2009), suggesting that STAT4 gene polymorphisms may contribute to RA susceptibility across ethnic barriers (Lee et al., 2010; Ji et al., 2010). However, the recent study between SNP rs11889341 in STAT4 gene and RA only found association of the heterozygous CT genotype with female RA group in Southwestern Chinese Han population, and there was no significant association between rs11889341 and the total patients (Li et al., 2009). A followed replication failure was also reported in African Americans (Kelley et al., 2010). The two inconsistent results make STAT4 gene now controversial. Thus, we undertook this study to investigate whether the association of STAT4 genetic polymorphisms with RA is present in Northwestern Chinese Han population. Materials and methods Subjects
RA patients. Serum levels of RF were tested by laser nephelometry. Individuals with values ≥40 IU/ml were regarded as RF positive. Serum anti-CCP antibody levels were examined by a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Immunoscan RA2, Generic Assays, Dahlewitz, Germany). A cut-off of 50 IU/ ml was used as a criterion for anti-CCP antibody positivity. Genotyping Genomic DNA was extracted from EDTA-anticoagulated peripheral blood using standard methods (Blin and Stafford, 1976). All DNA samples were stored at − 20 °C until analyzed. The four SNPs (rs7574865, rs8179673, rs10181656, rs11889341) in intron 3 of STAT4 gene were amplified by polymerase chain reaction (PCR) followed by denaturing high-performance liquid chromatography (DHPLC) on a Wave DNA Fragment Analysis System (Trans-gnomic Inc., San Jose. CA) as previously reported (Gross et al., 1999). The reference sequence (i.e. wild type sequence) of STAT4 gene was obtained from NCBI GenBank (NM_003151.2). The primer sequences for the four SNPs and the respectively melting temperature on PCR and DHPLC are listed in Supplementary table S1. Genotyping was conducted by laboratory personnel who were blinded to subject status, and all of the samples were randomly selected to be genotyped again by a different author. To confirm the genotyping results by PCR-DHPLC, DNA samples were also randomly selected to be examined again by direct sequencing on an automated ABI PRISM 3730 Genetic Analyzer (Sheng Gong Ltd., Shanghai, China) (sequencing more than 10% of the total sample).
This study was approved by the Ethical Committee of Lanzhou University. After obtaining informed consent, 208 RA patients (68 males, 140 females) and 312 healthy controls (119 males, 193 females) were consecutively recruited from the First and the Second Hospital of Lanzhou University, respectively, the People's Hospital of Gansu Province, the Traditional Chinese Medicine hospital of Gansu Province, and the affiliated Lanzhou Petrochemical hospital between July 2006 and November 2008. All patients, who self-reported Han Chinese, have resided in Northwestern China for at least three generations and fulfilled the revised criteria for the classification of rheumatoid arthritis by American Rheumatism Association in 1987 (Arnett et al., 1988), and their families did not have any record of RA history. The controls were gender, age and ethnically-matched unrelated healthy people obtained from the checkup population in the above five hospitals (Table 1).
Hardy–Weinberg equilibrium was assessed by using χ 2 test. Comparisons of allele and genotype frequencies between RA patients and controls were performed by using 2 × 2 contingency tables with χ 2 analysis. Haplotype constructions were analyzed by using SHEsis software (Shi and He, 2005). Multiple testing was corrected by Bonferroni procedure (Benjamini et al., 2001). All P-values were twotailed and P b 0.05 was considered to be statistically significant. All statistical analyses were performed with SPSS for Windows (version 16.0; SPSS Inc., Chicago, Illinois, USA).
Autoantibody analysis
Distribution of the four polymorphisms in cases and controls
The status of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibodies was available for about two-thirds of
The four SNPs have been successfully genotyped in all subjects, and repeated DHPLC results and corresponding sequencing results were 100% concordant (Supplementary Figure S1). All SNPs were in Hardy– Weinberg equilibrium in both patients and controls, and were significantly associated with RA (P b 0.05) in Northwestern Chinese Han population, with rs7574865 exhibiting the strongest association (uncorrected P = 1 × 10− 4, Odds Ratio (OR)= 1.645, 95% confidence interval (95% CI) = 1.272–2.129 for T vs. G; uncorrected P = 4.8 × 10 − 5, OR= 3.111, 95% CI = 1.777–5.447 for TT vs. GG). Furthermore, the associations remained significant except for rs11889341 after being corrected by Bonferroni procedure (Table 2).
Table 1 Characteristics of RA patients and controls. Characteristic
RA patients
Controls
Total number Female Male Age, mean ± SD years Age of onset, years Duration of disease, years RF+c RF−c Anti-CCP+c Anti-CCP−c
208 140 (67.31) 68 (32.69) 46.74 ± 16.44 40.7 ± 11.6 10.1 ± 9.3 87 (59.2) 60 (40.8) 71 (51.2) 68 (48.9)
312 193 (61.86) 119 (38.14) 47.04 ± 13.37
P value 0.205a N 0.05b
Values are numbers (%); RA, Rheumatoid Arthritis; RF, Rheumatoid factor; anti-CCP, anti-cyclic citrullinated peptide. a P value calculated by Pearson chi-square test (All frequency N0.05) or Fisher's exact test (Any frequency b0.05). b P value calculated by student t test. c Clinical data were not available for some cases.
Statistical analysis
Results
Stratified analysis of rs7574865 in subgroups according to the presence of RF and anti-CCP antibodies Since SNP rs7574865 exhibited the strongest association with RA in the present study, we analyzed the allele and genotype frequencies of rs7574865 among RA patients stratified by autoantibody status (Table 3). The stratification study showed that rs7574865 T allele was significantly associated with RF-positive and anti-CCP-positive subgroups (uncorrected P = 0.037, OR = 1.446, 95% CI = 1.021–2.047;
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Table 2 Genotype/allele frequencies of the four SNPs in STAT4 gene in RA patients and controls. SNPs
rs11889341
rs7574865
rs8179673
rs10181656
Genotype/Allele
P valuea
Genotype/Allele frequency
CC CT TT C T GG GT TT G T TT CT CC T C CC CG GG C G
Controls (n = 312)
Cases (n = 208)
133 145 34 411 213 144 141 27 429 195 136 138 38 410 214 151 140 21 442 182
76 95 37 247 169 72 94 42 238 178 67 106 35 240 176 77 105 26 259 157
(42.6) (46.5) (10.9) (65.9) (34.1) (46.2) (45.2) (8.7) (68.8) (31.2) (43.6) (44.2) (12.2) (65.7) (34.3) (48.4) (44.9) (6.7) (70.8) (29.2)
(36.5) (45.7) (17.8) (59.4) (40.6) (34.6) (45.2) (20.2) (57.2) (42.8) (32.2) (51.0) (16.8) (57.7) (42.3) (37.0) (50.5) (12.5) (62.3) (37.7)
OR (95% CI)
1 1.147 1.904 1 1.320 1 1.333 3.111 1 1.645 1 1.559 1.870 1 1.405 1 1.471 2.428 1 1.472
0.483 0.019 0.033 0.143 4.8 × 10− 5 1 × 10− 4 0.024 0.023 0.009 0.042 0.005 0.004
Pcorr
(0.782–1.681) (1.105–3.282)
0.076
(1.022–1.706)
0.132
(0.907–1.959) (1.777–5.447)
1.9 × 10− 4
(1.272–2.129)
4 × 10− 4
(1.059–2.296) (1.085–3.223)
0.096 0.092
(1.088–1.814)
0.036
(1.013–2.136) (1.284–4.592)
0.168 0.020
(1.132–1.915)
0.016
Values are number (%); MAF = minor allele frequency; OR, odds ratio; 95% CI = 95% confidence interval; Pcorr (corrected P) calculated by Bonferroni procedure. a P-values calculated by Pearson chi-square test.
uncorrected P = 0.003, OR= 1.754, 95% CI = 1.210–2.545, respectively) and showed a trend for association with RF-negative and anti-CCPnegative subgroups (uncorrected P = 0.061, OR = 1.467, 95% CI= 0.981–2.194; uncorrected P = 0.057, OR = 1.449, 95% CI = 1.449– 2.125, respectively). And the homozygous TT genotype of rs7574865 was strongly associated with not only RF-positive and anti-CCP-positive subgroups (uncorrected P = 0.011, OR = 2.510, 95% CI= 1.219–5.169 for RF-positive; P = 0.001, OR = 3.478, 95%CI = 1.611–7.508 for antiCCP-positive) but also with RF-negative and anti-CCP-negative subgroups (uncorrected P = 0.038, OR = 2.424, 95% CI= 1.033–5.689 for RF-negative; uncorrected P = 0.024, OR = 2.462, 95% CI= 1.108–5.467 for anti-CCP-negative). However, after being corrected by Bonferroni procedure, we only found significant associations of rs7574865 T allele and TT genotype with anti-CCP-positive subgroup (corrected P = 0.012 and 0.004, respectively), and a marginal association of rs7574865 TT genotype with RF-positive subgroup (corrected P = 0.044, Table 3). STAT4 haplotype analysis Linkage analysis of the four SNPs in STAT4 gene in controls showed moderate linkage disequilibrium (Fig. 1). Main haplotypes formed by rs11889341, rs7574865, rs8179673, and rs10181656 were shown in Table 4 (haplotypes with frequencies N3% were selected for analysis). The most common haplotype (CGTC) which was formed by the major allele of each SNP showed a protective effect to all RA patients (uncorrected P = 1.8 × 10 − 7, OR = 0.471, 95% CI = 0.354–0.626). While the second common haplotype (TTCG), formed by the minor
allele of each SNP, only showed a moderate risk effect for all the patients (uncorrected P = 0.049, OR = 1.394, 95% CI = 1.001–1.942). Furthermore, another risk haplotype (CTTC), which was formed by the minor allele of rs7574865 and the major alleles of the others was found to be significantly associated with RA in Northwestern Chinese Han population (uncorrected P = 1.9 × 10 − 6, OR = 2.966, 95% CI = 1.866–4.715). And after Bonferroni correction, the haplotypes CGTC and CTTC were still significantly associated with RA susceptibility (corrected P = 1.4 × 10 − 6 and 1.5 × 10 − 5, respectively). Discussion In the present study, we investigated the association of a candidate gene STAT4 with RA in a Chinese population. The four examined SNPs in intron 3 of STAT4 gene were associated with 1.3–1.6-fold increased susceptibility to Northwestern Chinese Han RA patients. These findings strongly support the hypothesis that STAT4 gene polymorphisms are also associated with RA in Northwestern Chinese Han population, and are similar to the results collected from major populations worldwide (Remmers et al., 2007; Lee et al., 2007; Palomino-Morales et al., 2008; Barton et al., 2008; Kobayashi et al., 2008; Orozco et al., 2008; Zervou et al., 2008; Martinez et al., 2008; Daha et al., 2009; Suarez-Gestal et al., 2009). Being a complex disease, clinical heterogeneity of RA may come from many factors such as the absence or presence of RF and anti-CCP antibodies. Our stratification study after Bonferroni correction showed that rs7574865 T allele and TT genotype were still
Table 3 Genotype frequencies of rs7574865 in RA patients, stratified by rheumatoid factor and anti-CCP autoantibody status. Groups
Genotype frequency GG
Control (n = 312) RF+ (n = 87)a RF− (n = 60)a Anti-CCP+ (n = 71)a Anti-CCP− (n = 68)a
144 34 22 23 26
(46.2) (39.1) (36.7) (32.4) (38.2)
T vs. G
GT
TT
141 (45.2) 37 (42.5) 28 (46.7) 33 (46.5) 30 (44.1)
27 16 10 15 12
(8.7) (18.4) (16.7) (21.1) (17.6)
TT vs. GG
P
OR (95% CI)
0.037 0.061 0.003 0.057
1 1.446 1.467 1.754 1.449
(1.021–2.047) (0.981–2.194) (1.210–2.545) (0.988–2.125)
Pcorr 0.148 0.244 0.012 0.228
P
OR (95% CI)
Pcorr
0.011 0.038 0.001 0.024
1 2.510 2.424 3.478 2.462
0.044 0.152 0.004 0.096
(1.219–5.169) (1.033–5.689) (1.611–7.508) (1.108–5.467)
Values are number (%) of genotypes; P-values calculated by Pearson chi-square test; Pcorr (corrected P) calculated by Bonferroni procedure; The odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated in the indicated patient subgroups in comparison to controls. a Clinical data were not available for some cases.
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Fig. 1. Pairwise linkage disequilibrium values of the single-nucleotide polymorphisms (SNPs) in intron 3 of STAT4 gene in the control samples.
significantly associated with anti-CCP-positive subgroup, however, only marginal association of TT genotype with RF-positive subgroup was observed, and no association was observed with anti-CCPnegative subgroup. These data suggest that STAT4 gene polymorphisms are associated with increased risk of antibody production to citrullinated proteins, as other genetic association studies revealed (Lee et al., 2007; Harrison et al., 2006). In our study, the previously reported (Remmers et al., 2007; Lee et al., 2007) protective haplotype (CGTC) also showed a significant association with RA in the Northwestern Chinese Han population, however, the previously reported risk haplotype (TTCG) only showed a marginal association with RA patients. Interestingly, another risk haplotype (CTTC) formed by the minor allele of rs7574865 and the major allele of the other three variants was found to be significantly associated with RA susceptibility. But, the frequency of this haplotype (CTTC) was a little lower in the controls (0.056) in this study, unfortunately, previously replication studies were mostly focused on the association of only rs7574865 polymorphism with RA (PalominoMorales et al., 2008; Kobayashi et al., 2008; Orozco et al., 2008; Zervou et al., 2008; Martinez et al., 2008; Daha et al., 2009; Suarez-Gestal et al., 2009). Therefore, this haplotype needs to be verified by a larger sample study in the future. STAT4, a latent cytosolic factor, has been the most extensively studied in helper T (Th) cells. IL-12 and IL-23 are key cytokines to activate STAT4 and then to direct Th cells toward the proinflammatory T-helper type 1 (Th1) and T-helper type 17 (Th17) linkages (Watford et al., 2004; Nishikomori et al., 2002). Variants or genetic differences of STAT4 may be involved in regulating the balance of IL-12 vs. IL-23 effect, and then affect the prevalence of inflammatory diseases via dysregulation of Th17 and Th1 activation. It has been reported that STAT4 was over expressed in RA synovial tissues than in normal tissues (Walker et al., 2006). Studies in mouse models of infectious and inflammatory diseases have revealed that STAT4 is the very important factor in mediating inflammatory immunity (Kaplan, 2005). Transgenic STAT4 knockout mice have been reported to have impaired Th1 differentiation, interferon-γ production, and cell-mediated immunity and are very susceptible to
intracellular infections (Watford et al., 2004), but they have less severe disease in proteoglycan-induced arthritis (Finnegan et al., 2002). And collagen-induced arthritis in mice could be ameliorated by antisense oligonucleotides directed at STAT4 (Hildner et al., 2007; Klinman et al., 2005; Shirota et al., 2004). All these suggest that STAT4 may play pivotal roles in both initiation and maintenance of inflammatory process and may be a potential therapeutic target for RA. Until now, two alternatively spliced isoforms have been described for STAT4, STAT4α and STAT4β, STAT4β which lacks 44 amino acids at the C terminus of the full-length STAT4α is not as efficient as STAT4α in directly inducing IFN-γ gene expression activated by IL-12 in Th1 cells (Hoey et al., 2003). A recent study has documented that STAT4 mRNA was over-expressed in osteoblasts with a risk haplotype formed by rs10181656 and rs7582694 (the two strongest SLEassociated SNPs) compared with osteoblasts with non-risk haplotypes (Sigurdsson et al., 2008). The expression level of STAT4 in Peripheral Blood Mononuclear Cell (PBMC) was also reported to be correlated with the risk allele of STAT4 rs7574865 (Abelson et al., 2009). Since the four susceptibility SNPs are located within the third intron of STAT4 gene, they probably have an influence on the level of STAT4 transcription and splice variation (Korman et al., 2008). However, the precisely functional roles of these risk SNPs remain to be elucidated. Conclusion In brief, our results confirm a genetic association of STAT4 with RA in Northwestern Chinese Han population, which strongly support the hypothesis that STAT4 may be a common RA susceptibility marker across different ethnic groups. Further functional studies on polymorphisms of STAT4 gene would lead to a greater understanding of RA and make it possible to develop novel therapies. Supplementary materials related to this article can be found online at doi:10.1016/j.lfs.2011.05.012. Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgments This work was funded by platform of Genetic Resource of Chinese Population Project, Ministry of Science and Technology of P. R. China (Grant No. 2006DKA21301), grant of the National Natural Science Foundation of China (Grant No. 30972708) and the National Science Foundation of China (Grant No. 81071701). We also gratefully acknowledge the numerous sample donors for making this work possible. References
Table 4 The frequencies of STAT4 haplotypes in all RA patients and controls. Haplotype
TGCG CGTC TTCG TGTC CTTC
Genotype/Allele frequency Controls (n = 312)
Cases (n = 208)
0.064 0.529 0.179 0.036 0.056
0.086 0.338 0.216 0.039 0.138
P valuea
OR (95% CI)
0.102 1.8 × 10− 7 0.049 0.635 1.9 × 10− 6
1.507 0.471 1.394 1.179 2.966
(0.920–2.469) (0.354–0.626) (1.001–1.942) (0.597–2.326) (1.866–4.715)
Pcorr
1.4 × 10− 6 0.392 1.5 × 10− 5
Haplotype block consists of SNPs: rs11889341, rs7574865, rs8179673, and rs10181656; Frequencies less than 0.03 in both RA and controls have been dropped; OR, odds ratio; CI, confidence interval; Pcorr (corrected P) calculated by Bonferroni multiple correct procedure. a P-values calculated by Pearson chi-square or Fisher's exact test.
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Signal transducer and activator of transcription 4 gene polymorphisms associated with rheumatoid arthritis in Northwestern Chinese Han population Ya-ling Liang a, Hua Wu a, Pei-qiang Li a, Xiao-dong Xie a,⁎, Xi Shen a, Xiao-qing Yang b, Xuan Cheng c, Li Liang d a
Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou City, 730000, Gansu Province, China Department of internal medicine, the affiliated Lanzhou Petrochemical hospital, Lanzhou City, 730060, Gansu Province, China Department of Laboratory Medicine, Traditional Chinese Medicine hospital of Gansu Province, Lanzhou City, 730050, Gansu Province, China d Department of Pharmacy, the first hospital of Lanzhou University, Lanzhou City, 730000, Gansu Province, China b c
a r t i c l e
i n f o
Article history: Received 4 October 2010 Accepted 19 May 2011 Keywords: STAT4 Rheumatoid arthritis Single nucleotide polymorphisms Case–control study
a b s t r a c t Aims: Signal transducer and activator of transcription 4 (STAT4) gene encode a transcriptional factor that transmits signals induced by several key cytokines which play important roles in the development of autoimmune diseases. Recently, several single nucleotide polymorphisms (SNPs) in STAT4 gene have been reported to be significantly associated with Rheumatoid arthritis (RA) in different ethnic populations. We undertook this study to investigate whether the association of STAT4 genetic polymorphisms with RA is present in Northwestern Chinese Han population. Main methods: A case–control association study in individuals with RA (n = 208) and healthy controls (n = 312) was conducted. Four SNPs (rs7574865, rs8179673, rs10181656, rs11889341) in STAT4 gene were genotyped by using polymerase chain reaction followed by denaturing high-performance liquid chromatography (PCR-DHPLC) and DNA sequencing. Key findings: The genotype and allele distributions of four polymorphisms were significantly different in individuals with RA compared to controls, with SNP rs7574865 T allele and T/T genotype showing the most significant association with susceptibility to RA (uncorrected P = 1 × 10− 4, OR = 1.645, 95% CI = 1.272–2.129; uncorrected P = 4.8 × 10− 5, OR = 3.111, 95% CI = 1.777–5.447, respectively). Stratification studies showed that STAT4 gene polymorphisms were significantly associated with anti-cyclic citrullinated peptide (antiCCP) positive subgroup in Northwestern Chinese Han population. Significance: These findings strongly suggest that STAT4 genetic polymorphisms are associated with RA in Northwestern Chinese Han population, and support the hypothesis of STAT4 gene polymorphisms increasing the risk for RA across major populations. © 2011 Elsevier Inc. All rights reserved.
Introduction Rheumatoid arthritis (RA) is a common chronic inflammatory autoimmune disease characterized by significant disability and early mortality, which affects ~1% of the adult population worldwide (Silman and Pearson, 2002). It is generally accepted that RA is a complex disease, with suspected interrelated contributions from genetic, infectious, environmental and hormonal factors (Firestein, 2003). Twin and family studies suggest that genetic factors contribute up to 60% of disease susceptibility to RA (MacGregor et al., 2000). To date, human leukocyte antigen (HLA) class II molecules have been documented as the most powerful genetic factors of RA across all populations, but they account for no more than one-third of the total
⁎ Corresponding author. Tel.: + 86 931 8915023; fax: + 86 931 8915001. E-mail address: [email protected] (X. Xie). 0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.05.012
genetic susceptibility (Newton et al., 2004; Cornelis et al., 1998). Recently, several non-HLA genes have been identified to contribute to RA susceptibility (Bowes and Barton, 2008). However, subsequent studies in European and Asian populations have got conflicting results (Yamada and Yamamoto, 2007; Kyogoku et al., 2004; Ikari et al., 2006; Orozco et al., 2005), which suggest that the genetic backgrounds of different ethnic groups should be taken into account (Colhoun et al., 2003). Signal transducer and activator of transcription 4 (STAT4) gene encode a transcription factor which resides in cytosol and transmits the intracellular signals induced by cytokines including interleukin-12 (IL-12), IL-23, IL-27 and type 1 interferons (IFNs). Upon cytokine signaling, this transcription factor becomes phosphorylated and translocates to the nucleus to play an essential downstream role in the differentiation and proliferation of IL-12-dependent T helper 1 (Th1) cells (Watford et al., 2004). STAT4 is also important in the development of IL-17-secreting Th cells (Th17) in response to IL-23 (Mathur et al., 2007). Since Th1 and Th17 lineages work as crucial
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effectors in chronic inflammatory disorders, STAT4 gene may play an important role in the pathogenesis of RA (McInnes and Schett, 2007; Thierfelder et al., 1996). Currently, several single nucleotide polymorphisms (SNPs) in STAT4 gene have been reported to be significantly associated with RA and systemic lupus erythematosus (SLE) (Amos et al., 2008; Remmers et al., 2007). And subsequent association studies between STAT4 gene polymorphisms and RA in Caucasians and Asians have yielded consistent results (Lee et al., 2007; Palomino-Morales et al., 2008; Barton et al., 2008; Kobayashi et al., 2008; Orozco et al., 2008; Martinez et al., 2008; Daha et al., 2009; Suarez-Gestal et al., 2009), suggesting that STAT4 gene polymorphisms may contribute to RA susceptibility across ethnic barriers (Lee et al., 2010; Ji et al., 2010). However, the recent study between SNP rs11889341 in STAT4 gene and RA only found association of the heterozygous CT genotype with female RA group in Southwestern Chinese Han population, and there was no significant association between rs11889341 and the total patients (Li et al., 2009). A followed replication failure was also reported in African Americans (Kelley et al., 2010). The two inconsistent results make STAT4 gene now controversial. Thus, we undertook this study to investigate whether the association of STAT4 genetic polymorphisms with RA is present in Northwestern Chinese Han population. Materials and methods Subjects
RA patients. Serum levels of RF were tested by laser nephelometry. Individuals with values ≥40 IU/ml were regarded as RF positive. Serum anti-CCP antibody levels were examined by a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Immunoscan RA2, Generic Assays, Dahlewitz, Germany). A cut-off of 50 IU/ ml was used as a criterion for anti-CCP antibody positivity. Genotyping Genomic DNA was extracted from EDTA-anticoagulated peripheral blood using standard methods (Blin and Stafford, 1976). All DNA samples were stored at − 20 °C until analyzed. The four SNPs (rs7574865, rs8179673, rs10181656, rs11889341) in intron 3 of STAT4 gene were amplified by polymerase chain reaction (PCR) followed by denaturing high-performance liquid chromatography (DHPLC) on a Wave DNA Fragment Analysis System (Trans-gnomic Inc., San Jose. CA) as previously reported (Gross et al., 1999). The reference sequence (i.e. wild type sequence) of STAT4 gene was obtained from NCBI GenBank (NM_003151.2). The primer sequences for the four SNPs and the respectively melting temperature on PCR and DHPLC are listed in Supplementary table S1. Genotyping was conducted by laboratory personnel who were blinded to subject status, and all of the samples were randomly selected to be genotyped again by a different author. To confirm the genotyping results by PCR-DHPLC, DNA samples were also randomly selected to be examined again by direct sequencing on an automated ABI PRISM 3730 Genetic Analyzer (Sheng Gong Ltd., Shanghai, China) (sequencing more than 10% of the total sample).
This study was approved by the Ethical Committee of Lanzhou University. After obtaining informed consent, 208 RA patients (68 males, 140 females) and 312 healthy controls (119 males, 193 females) were consecutively recruited from the First and the Second Hospital of Lanzhou University, respectively, the People's Hospital of Gansu Province, the Traditional Chinese Medicine hospital of Gansu Province, and the affiliated Lanzhou Petrochemical hospital between July 2006 and November 2008. All patients, who self-reported Han Chinese, have resided in Northwestern China for at least three generations and fulfilled the revised criteria for the classification of rheumatoid arthritis by American Rheumatism Association in 1987 (Arnett et al., 1988), and their families did not have any record of RA history. The controls were gender, age and ethnically-matched unrelated healthy people obtained from the checkup population in the above five hospitals (Table 1).
Hardy–Weinberg equilibrium was assessed by using χ 2 test. Comparisons of allele and genotype frequencies between RA patients and controls were performed by using 2 × 2 contingency tables with χ 2 analysis. Haplotype constructions were analyzed by using SHEsis software (Shi and He, 2005). Multiple testing was corrected by Bonferroni procedure (Benjamini et al., 2001). All P-values were twotailed and P b 0.05 was considered to be statistically significant. All statistical analyses were performed with SPSS for Windows (version 16.0; SPSS Inc., Chicago, Illinois, USA).
Autoantibody analysis
Distribution of the four polymorphisms in cases and controls
The status of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibodies was available for about two-thirds of
The four SNPs have been successfully genotyped in all subjects, and repeated DHPLC results and corresponding sequencing results were 100% concordant (Supplementary Figure S1). All SNPs were in Hardy– Weinberg equilibrium in both patients and controls, and were significantly associated with RA (P b 0.05) in Northwestern Chinese Han population, with rs7574865 exhibiting the strongest association (uncorrected P = 1 × 10− 4, Odds Ratio (OR)= 1.645, 95% confidence interval (95% CI) = 1.272–2.129 for T vs. G; uncorrected P = 4.8 × 10 − 5, OR= 3.111, 95% CI = 1.777–5.447 for TT vs. GG). Furthermore, the associations remained significant except for rs11889341 after being corrected by Bonferroni procedure (Table 2).
Table 1 Characteristics of RA patients and controls. Characteristic
RA patients
Controls
Total number Female Male Age, mean ± SD years Age of onset, years Duration of disease, years RF+c RF−c Anti-CCP+c Anti-CCP−c
208 140 (67.31) 68 (32.69) 46.74 ± 16.44 40.7 ± 11.6 10.1 ± 9.3 87 (59.2) 60 (40.8) 71 (51.2) 68 (48.9)
312 193 (61.86) 119 (38.14) 47.04 ± 13.37
P value 0.205a N 0.05b
Values are numbers (%); RA, Rheumatoid Arthritis; RF, Rheumatoid factor; anti-CCP, anti-cyclic citrullinated peptide. a P value calculated by Pearson chi-square test (All frequency N0.05) or Fisher's exact test (Any frequency b0.05). b P value calculated by student t test. c Clinical data were not available for some cases.
Statistical analysis
Results
Stratified analysis of rs7574865 in subgroups according to the presence of RF and anti-CCP antibodies Since SNP rs7574865 exhibited the strongest association with RA in the present study, we analyzed the allele and genotype frequencies of rs7574865 among RA patients stratified by autoantibody status (Table 3). The stratification study showed that rs7574865 T allele was significantly associated with RF-positive and anti-CCP-positive subgroups (uncorrected P = 0.037, OR = 1.446, 95% CI = 1.021–2.047;
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Table 2 Genotype/allele frequencies of the four SNPs in STAT4 gene in RA patients and controls. SNPs
rs11889341
rs7574865
rs8179673
rs10181656
Genotype/Allele
P valuea
Genotype/Allele frequency
CC CT TT C T GG GT TT G T TT CT CC T C CC CG GG C G
Controls (n = 312)
Cases (n = 208)
133 145 34 411 213 144 141 27 429 195 136 138 38 410 214 151 140 21 442 182
76 95 37 247 169 72 94 42 238 178 67 106 35 240 176 77 105 26 259 157
(42.6) (46.5) (10.9) (65.9) (34.1) (46.2) (45.2) (8.7) (68.8) (31.2) (43.6) (44.2) (12.2) (65.7) (34.3) (48.4) (44.9) (6.7) (70.8) (29.2)
(36.5) (45.7) (17.8) (59.4) (40.6) (34.6) (45.2) (20.2) (57.2) (42.8) (32.2) (51.0) (16.8) (57.7) (42.3) (37.0) (50.5) (12.5) (62.3) (37.7)
OR (95% CI)
1 1.147 1.904 1 1.320 1 1.333 3.111 1 1.645 1 1.559 1.870 1 1.405 1 1.471 2.428 1 1.472
0.483 0.019 0.033 0.143 4.8 × 10− 5 1 × 10− 4 0.024 0.023 0.009 0.042 0.005 0.004
Pcorr
(0.782–1.681) (1.105–3.282)
0.076
(1.022–1.706)
0.132
(0.907–1.959) (1.777–5.447)
1.9 × 10− 4
(1.272–2.129)
4 × 10− 4
(1.059–2.296) (1.085–3.223)
0.096 0.092
(1.088–1.814)
0.036
(1.013–2.136) (1.284–4.592)
0.168 0.020
(1.132–1.915)
0.016
Values are number (%); MAF = minor allele frequency; OR, odds ratio; 95% CI = 95% confidence interval; Pcorr (corrected P) calculated by Bonferroni procedure. a P-values calculated by Pearson chi-square test.
uncorrected P = 0.003, OR= 1.754, 95% CI = 1.210–2.545, respectively) and showed a trend for association with RF-negative and anti-CCPnegative subgroups (uncorrected P = 0.061, OR = 1.467, 95% CI= 0.981–2.194; uncorrected P = 0.057, OR = 1.449, 95% CI = 1.449– 2.125, respectively). And the homozygous TT genotype of rs7574865 was strongly associated with not only RF-positive and anti-CCP-positive subgroups (uncorrected P = 0.011, OR = 2.510, 95% CI= 1.219–5.169 for RF-positive; P = 0.001, OR = 3.478, 95%CI = 1.611–7.508 for antiCCP-positive) but also with RF-negative and anti-CCP-negative subgroups (uncorrected P = 0.038, OR = 2.424, 95% CI= 1.033–5.689 for RF-negative; uncorrected P = 0.024, OR = 2.462, 95% CI= 1.108–5.467 for anti-CCP-negative). However, after being corrected by Bonferroni procedure, we only found significant associations of rs7574865 T allele and TT genotype with anti-CCP-positive subgroup (corrected P = 0.012 and 0.004, respectively), and a marginal association of rs7574865 TT genotype with RF-positive subgroup (corrected P = 0.044, Table 3). STAT4 haplotype analysis Linkage analysis of the four SNPs in STAT4 gene in controls showed moderate linkage disequilibrium (Fig. 1). Main haplotypes formed by rs11889341, rs7574865, rs8179673, and rs10181656 were shown in Table 4 (haplotypes with frequencies N3% were selected for analysis). The most common haplotype (CGTC) which was formed by the major allele of each SNP showed a protective effect to all RA patients (uncorrected P = 1.8 × 10 − 7, OR = 0.471, 95% CI = 0.354–0.626). While the second common haplotype (TTCG), formed by the minor
allele of each SNP, only showed a moderate risk effect for all the patients (uncorrected P = 0.049, OR = 1.394, 95% CI = 1.001–1.942). Furthermore, another risk haplotype (CTTC), which was formed by the minor allele of rs7574865 and the major alleles of the others was found to be significantly associated with RA in Northwestern Chinese Han population (uncorrected P = 1.9 × 10 − 6, OR = 2.966, 95% CI = 1.866–4.715). And after Bonferroni correction, the haplotypes CGTC and CTTC were still significantly associated with RA susceptibility (corrected P = 1.4 × 10 − 6 and 1.5 × 10 − 5, respectively). Discussion In the present study, we investigated the association of a candidate gene STAT4 with RA in a Chinese population. The four examined SNPs in intron 3 of STAT4 gene were associated with 1.3–1.6-fold increased susceptibility to Northwestern Chinese Han RA patients. These findings strongly support the hypothesis that STAT4 gene polymorphisms are also associated with RA in Northwestern Chinese Han population, and are similar to the results collected from major populations worldwide (Remmers et al., 2007; Lee et al., 2007; Palomino-Morales et al., 2008; Barton et al., 2008; Kobayashi et al., 2008; Orozco et al., 2008; Zervou et al., 2008; Martinez et al., 2008; Daha et al., 2009; Suarez-Gestal et al., 2009). Being a complex disease, clinical heterogeneity of RA may come from many factors such as the absence or presence of RF and anti-CCP antibodies. Our stratification study after Bonferroni correction showed that rs7574865 T allele and TT genotype were still
Table 3 Genotype frequencies of rs7574865 in RA patients, stratified by rheumatoid factor and anti-CCP autoantibody status. Groups
Genotype frequency GG
Control (n = 312) RF+ (n = 87)a RF− (n = 60)a Anti-CCP+ (n = 71)a Anti-CCP− (n = 68)a
144 34 22 23 26
(46.2) (39.1) (36.7) (32.4) (38.2)
T vs. G
GT
TT
141 (45.2) 37 (42.5) 28 (46.7) 33 (46.5) 30 (44.1)
27 16 10 15 12
(8.7) (18.4) (16.7) (21.1) (17.6)
TT vs. GG
P
OR (95% CI)
0.037 0.061 0.003 0.057
1 1.446 1.467 1.754 1.449
(1.021–2.047) (0.981–2.194) (1.210–2.545) (0.988–2.125)
Pcorr 0.148 0.244 0.012 0.228
P
OR (95% CI)
Pcorr
0.011 0.038 0.001 0.024
1 2.510 2.424 3.478 2.462
0.044 0.152 0.004 0.096
(1.219–5.169) (1.033–5.689) (1.611–7.508) (1.108–5.467)
Values are number (%) of genotypes; P-values calculated by Pearson chi-square test; Pcorr (corrected P) calculated by Bonferroni procedure; The odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated in the indicated patient subgroups in comparison to controls. a Clinical data were not available for some cases.
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Fig. 1. Pairwise linkage disequilibrium values of the single-nucleotide polymorphisms (SNPs) in intron 3 of STAT4 gene in the control samples.
significantly associated with anti-CCP-positive subgroup, however, only marginal association of TT genotype with RF-positive subgroup was observed, and no association was observed with anti-CCPnegative subgroup. These data suggest that STAT4 gene polymorphisms are associated with increased risk of antibody production to citrullinated proteins, as other genetic association studies revealed (Lee et al., 2007; Harrison et al., 2006). In our study, the previously reported (Remmers et al., 2007; Lee et al., 2007) protective haplotype (CGTC) also showed a significant association with RA in the Northwestern Chinese Han population, however, the previously reported risk haplotype (TTCG) only showed a marginal association with RA patients. Interestingly, another risk haplotype (CTTC) formed by the minor allele of rs7574865 and the major allele of the other three variants was found to be significantly associated with RA susceptibility. But, the frequency of this haplotype (CTTC) was a little lower in the controls (0.056) in this study, unfortunately, previously replication studies were mostly focused on the association of only rs7574865 polymorphism with RA (PalominoMorales et al., 2008; Kobayashi et al., 2008; Orozco et al., 2008; Zervou et al., 2008; Martinez et al., 2008; Daha et al., 2009; Suarez-Gestal et al., 2009). Therefore, this haplotype needs to be verified by a larger sample study in the future. STAT4, a latent cytosolic factor, has been the most extensively studied in helper T (Th) cells. IL-12 and IL-23 are key cytokines to activate STAT4 and then to direct Th cells toward the proinflammatory T-helper type 1 (Th1) and T-helper type 17 (Th17) linkages (Watford et al., 2004; Nishikomori et al., 2002). Variants or genetic differences of STAT4 may be involved in regulating the balance of IL-12 vs. IL-23 effect, and then affect the prevalence of inflammatory diseases via dysregulation of Th17 and Th1 activation. It has been reported that STAT4 was over expressed in RA synovial tissues than in normal tissues (Walker et al., 2006). Studies in mouse models of infectious and inflammatory diseases have revealed that STAT4 is the very important factor in mediating inflammatory immunity (Kaplan, 2005). Transgenic STAT4 knockout mice have been reported to have impaired Th1 differentiation, interferon-γ production, and cell-mediated immunity and are very susceptible to
intracellular infections (Watford et al., 2004), but they have less severe disease in proteoglycan-induced arthritis (Finnegan et al., 2002). And collagen-induced arthritis in mice could be ameliorated by antisense oligonucleotides directed at STAT4 (Hildner et al., 2007; Klinman et al., 2005; Shirota et al., 2004). All these suggest that STAT4 may play pivotal roles in both initiation and maintenance of inflammatory process and may be a potential therapeutic target for RA. Until now, two alternatively spliced isoforms have been described for STAT4, STAT4α and STAT4β, STAT4β which lacks 44 amino acids at the C terminus of the full-length STAT4α is not as efficient as STAT4α in directly inducing IFN-γ gene expression activated by IL-12 in Th1 cells (Hoey et al., 2003). A recent study has documented that STAT4 mRNA was over-expressed in osteoblasts with a risk haplotype formed by rs10181656 and rs7582694 (the two strongest SLEassociated SNPs) compared with osteoblasts with non-risk haplotypes (Sigurdsson et al., 2008). The expression level of STAT4 in Peripheral Blood Mononuclear Cell (PBMC) was also reported to be correlated with the risk allele of STAT4 rs7574865 (Abelson et al., 2009). Since the four susceptibility SNPs are located within the third intron of STAT4 gene, they probably have an influence on the level of STAT4 transcription and splice variation (Korman et al., 2008). However, the precisely functional roles of these risk SNPs remain to be elucidated. Conclusion In brief, our results confirm a genetic association of STAT4 with RA in Northwestern Chinese Han population, which strongly support the hypothesis that STAT4 may be a common RA susceptibility marker across different ethnic groups. Further functional studies on polymorphisms of STAT4 gene would lead to a greater understanding of RA and make it possible to develop novel therapies. Supplementary materials related to this article can be found online at doi:10.1016/j.lfs.2011.05.012. Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgments This work was funded by platform of Genetic Resource of Chinese Population Project, Ministry of Science and Technology of P. R. China (Grant No. 2006DKA21301), grant of the National Natural Science Foundation of China (Grant No. 30972708) and the National Science Foundation of China (Grant No. 81071701). We also gratefully acknowledge the numerous sample donors for making this work possible. References
Table 4 The frequencies of STAT4 haplotypes in all RA patients and controls. Haplotype
TGCG CGTC TTCG TGTC CTTC
Genotype/Allele frequency Controls (n = 312)
Cases (n = 208)
0.064 0.529 0.179 0.036 0.056
0.086 0.338 0.216 0.039 0.138
P valuea
OR (95% CI)
0.102 1.8 × 10− 7 0.049 0.635 1.9 × 10− 6
1.507 0.471 1.394 1.179 2.966
(0.920–2.469) (0.354–0.626) (1.001–1.942) (0.597–2.326) (1.866–4.715)
Pcorr
1.4 × 10− 6 0.392 1.5 × 10− 5
Haplotype block consists of SNPs: rs11889341, rs7574865, rs8179673, and rs10181656; Frequencies less than 0.03 in both RA and controls have been dropped; OR, odds ratio; CI, confidence interval; Pcorr (corrected P) calculated by Bonferroni multiple correct procedure. a P-values calculated by Pearson chi-square or Fisher's exact test.
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