doi: 10.1111/iji.12148
Polymorphisms in the TNF-a, TNFR1 gene and risk of rheumatoid arthritis in Chinese Han population F. Li*,†,1, J. Gao‡,1, J. Sokolove§, J. Xu¶, J. Zheng*, K. Zhu* & Z. Pan*,†
Summary Recent advances have highlighted a major genetic contribution to the pathogenesis of rheumatoid arthritis (RA).The aim of this study was to investigate whether polymorphisms of TNF-a (rs1800630, rs1800629) and TNFR1 (rs767455) were associated with susceptibility to and clinical outcome of RA in Chinese Han population. The target gene polymorphisms were genotyped in 256 patients with RA and 331 healthy controls using a high resolution melting (HRM) method. ESR, CRP, RF anti-CCP and anti-GPI level were also assayed and compared in genotypes of each polymorphism. Significant difference was observed in the genotype distributions and allele frequencies of TNF-a rs1800629 (P = 0.001, P < 0.001, respectively) between patients with RA and controls. There is no evidence to suggest an association between genotypes of the 3 SNPs according to age, gender, disease duration, DAS28 and serum level of autoantibodies. This study identifies a potentially important role for TNF-a rs1800629 polymorphisms in the susceptibility to RA.However, further studies in larger cohorts are required.
Introduction Rheumatoid arthritis (RA) is a common systemic autoimmune disease characterized by chronic inflammation
* Department of Rheumatology, Shandong University Qilu Hospital, Qingdao, Shandong, China, † Department of Rheumatology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, China, ‡ Department of Clinical Psycology, Shandong University Qilu Hospital, Qingdao, Shandong, China, § Division of Immunology and Rheumatology, Stanford University Medical Center, VA Palo Alto Health Care System, Palo Alto, CA, USA and ¶ Department of Rheumatology, Laiwu City People’s Hospital, Laiwu, Shandong, China Received 15 June 2014; revised 22 July 2014; accepted 6 August 2014 Correspondence: Zhenglun Pan, Department of Rheumatology, Shandong University Qilu Hospital , 758 Hefei Road, Qingdao, 266000, Shandong, China. Tel: 01186185 61812108; Fax: 0118653286580532; E-mail: [email protected] 1
Contribute equally with this project.
© 2014 John Wiley & Sons Ltd International Journal of Immunogenetics, 2014, 41, 499–502
of the synovial membrane in multiple joints. Both genetic and environmental factors play a role in the pathogenesis of the disease (Gabriel, 2001). Data from twin and familial studies have revealed that risk alleles from many different genes predispose to RA susceptibility (Silman et al., 1993). The association of the HLA-DR alleles is strongest and most widely recognized, but accounts for no more than 30% of the total genetic background, thus there are clearly additional genetic factors predisposing to RA (Kurko et al., 2013). Additionally, there are polymorphisms in several genetic loci which may contribute to the progression and severity of RA. Several cytokine-associated genes have been considered candidates for influencing disease susceptibility or severity (Harrison et al., 2008; Emonts et al., 2011). TNF-a is a potent immune mediator and proinflammatory cytokine that has been implicated in the pathogenesis of a large number of human diseases (Mosaad et al., 2011). Antitumour necrosis factor (anti-TNF) therapy is a breakthrough in the management of rheumatoid arthritis (Mugnier et al., 2003). High concentrations of TNF-a have been found in the plasma, synovial fluid and tissue of patients with RA (You et al., 2007), and the TNF-a polymorphism rs1800629 has been reported to be associated with several autoimmune diseases including RA (Al-Rayes et al., 2011). TNF-a exerts its biological activity by binding to the TNF receptors (TNFR) type I and II (Valle et al., 2010). TNFR1 is expressed on all nucleated cells, particularly those susceptible to the cytotoxic action of TNF and has been shown to be associated with susceptibility to RA, PsA and juvenile rheumatoid arthritis (Bridges et al., 2002; Miterski et al., 2004; Morales-Lara et al., 2012). In this study, we investigated a total of three polymorphisms in two genes to assess whether these polymorphisms could be a marker of susceptibility to or severity of RA. Polymorphisms evaluated included TNF-a gene (rs1800630, rs1800629) and TNFR1 (rs767455).The analysis was performed on a case–control cohort of Chinese Han population from Shandong Province. Additionally, we examined whether the association was restricted to clinically relevant disease subsets as characterized by autoantibody status.
499
500
F. Li et al.
Materials and methods Patients and controls
A total of 256-unrelated Chinese patients with RA whose diagnosis fulfilled the 2010 ACR-EULAR Classification Criteria were recruited in from the department of rheumatology, provincial hospital affiliated to Shandong University, China. For RA cohorts, RF, antiCCP2, anti-GPI, ESR and CRP status were obtained. As healthy controls, 331 age and sex matched healthy volunteers from the same geographic and ethnic background were included. All patients and controls gave their informed consent to participate in the study, all participants provided their written informed consent to participate, and the study was approved by the ethics committee of the provincial hospital affiliated to Shandong University. The characteristic of the participates was as follows: patients with RA (female 196, male 60; mean age 50.26 12.86 years), median disease duration 3 (0.8–8.0) years, healthy controls (female 239, male 92; mean age 48.08 13.92 years). RF was measured using N Latex RF Kit (SIEMENS, Berlin, Germany), anti-GPI and anti-CCP2 was determined by enzyme-linked immunosorbent assay (ELISA) (AESKULISA, Wendelsheim, Germany) . ESR and CRP were determined by the Westergren method via a Monitor-100 (Balzella, Forli, Italy) and the immune turbidimetric analysis (SIEMENS), respectively. Disease Activity Score in 28 joints for rheumatoid arthritis (DAS28) was calculated using a formula that includes the patients’ general/global health (measured on a Visual Analogue Scale of 100 mm), the number of tender joints and swollen joints (28 joints maximum) and the ESR or CRP (http://www.das-score.nl/). The mean score of DAS28 of the patients with RA in this study is 5.26 1.49 (DAS28 > 5.1 indicating high disease activity).
Statistical analysis
Analysis of the 3 SNPs allele and genotype frequencies showed that they were in Hardy–Weinberg equilibrium as examined by chi-square goodness-of-fit test using gene frequencies observed in healthy controls. The distribution of genotypes and allele frequency of patients with RA for the 3 SNPs were compared with control groups using the Pearson’s chi-square test or Fisher’s exact test as appropriate. The mean age was compared between RA and controls using Students t-test. Association between genotypes, age, disease duration, ESR, CRP, DAS28 and autoantibodies levels were assessed using the one-way ANOVA or Kruskal–Wallis one-way analysis of variance (ANOVA) if equal variances were not assumed. Where appropriate, adjustment for potential multiple testing errors was carried out, either by Bonferroni correction for normally distributed data or by the Kruskal–Wallis z-value test for nonparametric data. Linkage disequilibrium analysis was analysed by SHEsis http://analysis.bio-x.cn/myAnalysis.php). All data were analysed using R package (http://www.r-pro ject.org). P values less than 0.05 were considered to be significant.
Results Alleles, genotypes and susceptibility to RA
The polymorphisms TNF-a rs1800629 showed an apparent association with RA in the Chinese cohort. TNF-a rs1800629 was negatively associated with RA as summarized in Table 1. We observed that the AA + AG genotype of the TNF-a rs1800629 was significantly lower in patients with RA (3.9%) than in healthy controls (12.4%) (P = 0.001), and the allele rates in RA compared with controls was 2.0% versus 6.3% (P < 0.001).
Selection of candidate SNPs and sample genotyping
Subgroup and association of RA
The tag SNPs of TNF-a and TNFR1 were selected for their minor allele frequency (MAF) >10% in the Chinese population in the dbSNP database (http://www. ncbi.nlm.nih.gov/SNP/),and from results of previous published studies. Finally, we chose 3 candidate variants TNF-a (rs1800630, rs1800629) and TNFR1 (rs767455). Genomic DNA was extracted from blood samples collected in EDTA using a Relax Gene Blood DNA System (TIANGEN BIOTECH, Beijing, China) . The target polymorphisms were amplified by polymerase chain reaction (PCR) using TaKaRa Taq Hot Start Version (TaKaRa Biotechnology, Shiga, Japan) and genotyping by high resolution melting (HRM) methods using LightCycler480 (Roche, Basel, Switzerland). We randomly confirmed our genotyping results by direct sequencing by an ABI3730 sequencer. The details of the 3 SNPs can be seen in Table 1.
For a better understanding of disease aetiology, we investigated whether the observed associations were restricted to a specific subset of disease, defined by age, gender, mean disease duration, DAS28 and autoantibody status. In these subsets, no differential association could be observed for any of the 3 studied SNPs. Age was older in patients with the mutant homozygous genotype compared with heterozygous and wildtype homozygous, although this was not statistically significant. There was a trend of increasing disease duration, ESR, CRP, DAS28 and autoantibody status for some SNPs, but none were statistically significant (Tables 2 and 3).
Discussion In the present study, we have focused on polymorphisms with potential functional relevance or previ-
© 2014 John Wiley & Sons Ltd International Journal of Immunogenetics, 2014, 41, 499–502
TNF-a polymorphisms in the susceptibility to RA
501
Table 1. Genotype distributions and allele frequencies of the 3 SNPs in Chinese Han population from Shandong province Genotype Gene
SNP
Allele 1/2
TNF-a
rs1800630
C/A
rs1800629
G/A
rs767455
T/C
TNFR1
Group
11
RA Control RA Control RA Control Control
177 220 246 290 200 258 145
Allele 12
(69.1) (66.5) (96.1) (87.6) (78.1) (77.9) (43.8)
68 100 10 40 56 71 148
22 (26.6) (30.2) (3.9) (12.1) (21.9) (21.5) (44.7)
11 11 0 1 0 2 38
(4.3) (3.3) (0) (0.3) (0) ((0.6) (11.5)
P
1
0.55
422 540 502 620 456 587 438
0.001 0.46
2 (82.4) (81.6) (98.0) (93.7) (89.1) (88.7) (66.2)
90 122 10 42 56 75 224
(17.6) (18.4) (2.0) (6.3) (10.9) (11.3) (33.8)
P
OR
95% CI
1.06
0.78–1.43
0.76
3.4
1.69–6.85
C TNFRI polymorphism is associated with soluble levels and clinical activity in rheumatoid arthritis. Rheumatology International, 30, 655. You, C.G., Yin, Y.S., Xie, X.D., Ju, J., Wang, Z.P. & Chen, Y.R. (2007) Sex influences on the penetrance of IL-1beta and IL-1RN genotypes for rheumatoid arthritis in the Chinese population. Journal of International Medical Research, 35, 323.
© 2014 John Wiley & Sons Ltd International Journal of Immunogenetics, 2014, 41, 499–502
Polymorphisms in the TNF-a, TNFR1 gene and risk of rheumatoid arthritis in Chinese Han population F. Li*,†,1, J. Gao‡,1, J. Sokolove§, J. Xu¶, J. Zheng*, K. Zhu* & Z. Pan*,†
Summary Recent advances have highlighted a major genetic contribution to the pathogenesis of rheumatoid arthritis (RA).The aim of this study was to investigate whether polymorphisms of TNF-a (rs1800630, rs1800629) and TNFR1 (rs767455) were associated with susceptibility to and clinical outcome of RA in Chinese Han population. The target gene polymorphisms were genotyped in 256 patients with RA and 331 healthy controls using a high resolution melting (HRM) method. ESR, CRP, RF anti-CCP and anti-GPI level were also assayed and compared in genotypes of each polymorphism. Significant difference was observed in the genotype distributions and allele frequencies of TNF-a rs1800629 (P = 0.001, P < 0.001, respectively) between patients with RA and controls. There is no evidence to suggest an association between genotypes of the 3 SNPs according to age, gender, disease duration, DAS28 and serum level of autoantibodies. This study identifies a potentially important role for TNF-a rs1800629 polymorphisms in the susceptibility to RA.However, further studies in larger cohorts are required.
Introduction Rheumatoid arthritis (RA) is a common systemic autoimmune disease characterized by chronic inflammation
* Department of Rheumatology, Shandong University Qilu Hospital, Qingdao, Shandong, China, † Department of Rheumatology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, China, ‡ Department of Clinical Psycology, Shandong University Qilu Hospital, Qingdao, Shandong, China, § Division of Immunology and Rheumatology, Stanford University Medical Center, VA Palo Alto Health Care System, Palo Alto, CA, USA and ¶ Department of Rheumatology, Laiwu City People’s Hospital, Laiwu, Shandong, China Received 15 June 2014; revised 22 July 2014; accepted 6 August 2014 Correspondence: Zhenglun Pan, Department of Rheumatology, Shandong University Qilu Hospital , 758 Hefei Road, Qingdao, 266000, Shandong, China. Tel: 01186185 61812108; Fax: 0118653286580532; E-mail: [email protected] 1
Contribute equally with this project.
© 2014 John Wiley & Sons Ltd International Journal of Immunogenetics, 2014, 41, 499–502
of the synovial membrane in multiple joints. Both genetic and environmental factors play a role in the pathogenesis of the disease (Gabriel, 2001). Data from twin and familial studies have revealed that risk alleles from many different genes predispose to RA susceptibility (Silman et al., 1993). The association of the HLA-DR alleles is strongest and most widely recognized, but accounts for no more than 30% of the total genetic background, thus there are clearly additional genetic factors predisposing to RA (Kurko et al., 2013). Additionally, there are polymorphisms in several genetic loci which may contribute to the progression and severity of RA. Several cytokine-associated genes have been considered candidates for influencing disease susceptibility or severity (Harrison et al., 2008; Emonts et al., 2011). TNF-a is a potent immune mediator and proinflammatory cytokine that has been implicated in the pathogenesis of a large number of human diseases (Mosaad et al., 2011). Antitumour necrosis factor (anti-TNF) therapy is a breakthrough in the management of rheumatoid arthritis (Mugnier et al., 2003). High concentrations of TNF-a have been found in the plasma, synovial fluid and tissue of patients with RA (You et al., 2007), and the TNF-a polymorphism rs1800629 has been reported to be associated with several autoimmune diseases including RA (Al-Rayes et al., 2011). TNF-a exerts its biological activity by binding to the TNF receptors (TNFR) type I and II (Valle et al., 2010). TNFR1 is expressed on all nucleated cells, particularly those susceptible to the cytotoxic action of TNF and has been shown to be associated with susceptibility to RA, PsA and juvenile rheumatoid arthritis (Bridges et al., 2002; Miterski et al., 2004; Morales-Lara et al., 2012). In this study, we investigated a total of three polymorphisms in two genes to assess whether these polymorphisms could be a marker of susceptibility to or severity of RA. Polymorphisms evaluated included TNF-a gene (rs1800630, rs1800629) and TNFR1 (rs767455).The analysis was performed on a case–control cohort of Chinese Han population from Shandong Province. Additionally, we examined whether the association was restricted to clinically relevant disease subsets as characterized by autoantibody status.
499
500
F. Li et al.
Materials and methods Patients and controls
A total of 256-unrelated Chinese patients with RA whose diagnosis fulfilled the 2010 ACR-EULAR Classification Criteria were recruited in from the department of rheumatology, provincial hospital affiliated to Shandong University, China. For RA cohorts, RF, antiCCP2, anti-GPI, ESR and CRP status were obtained. As healthy controls, 331 age and sex matched healthy volunteers from the same geographic and ethnic background were included. All patients and controls gave their informed consent to participate in the study, all participants provided their written informed consent to participate, and the study was approved by the ethics committee of the provincial hospital affiliated to Shandong University. The characteristic of the participates was as follows: patients with RA (female 196, male 60; mean age 50.26 12.86 years), median disease duration 3 (0.8–8.0) years, healthy controls (female 239, male 92; mean age 48.08 13.92 years). RF was measured using N Latex RF Kit (SIEMENS, Berlin, Germany), anti-GPI and anti-CCP2 was determined by enzyme-linked immunosorbent assay (ELISA) (AESKULISA, Wendelsheim, Germany) . ESR and CRP were determined by the Westergren method via a Monitor-100 (Balzella, Forli, Italy) and the immune turbidimetric analysis (SIEMENS), respectively. Disease Activity Score in 28 joints for rheumatoid arthritis (DAS28) was calculated using a formula that includes the patients’ general/global health (measured on a Visual Analogue Scale of 100 mm), the number of tender joints and swollen joints (28 joints maximum) and the ESR or CRP (http://www.das-score.nl/). The mean score of DAS28 of the patients with RA in this study is 5.26 1.49 (DAS28 > 5.1 indicating high disease activity).
Statistical analysis
Analysis of the 3 SNPs allele and genotype frequencies showed that they were in Hardy–Weinberg equilibrium as examined by chi-square goodness-of-fit test using gene frequencies observed in healthy controls. The distribution of genotypes and allele frequency of patients with RA for the 3 SNPs were compared with control groups using the Pearson’s chi-square test or Fisher’s exact test as appropriate. The mean age was compared between RA and controls using Students t-test. Association between genotypes, age, disease duration, ESR, CRP, DAS28 and autoantibodies levels were assessed using the one-way ANOVA or Kruskal–Wallis one-way analysis of variance (ANOVA) if equal variances were not assumed. Where appropriate, adjustment for potential multiple testing errors was carried out, either by Bonferroni correction for normally distributed data or by the Kruskal–Wallis z-value test for nonparametric data. Linkage disequilibrium analysis was analysed by SHEsis http://analysis.bio-x.cn/myAnalysis.php). All data were analysed using R package (http://www.r-pro ject.org). P values less than 0.05 were considered to be significant.
Results Alleles, genotypes and susceptibility to RA
The polymorphisms TNF-a rs1800629 showed an apparent association with RA in the Chinese cohort. TNF-a rs1800629 was negatively associated with RA as summarized in Table 1. We observed that the AA + AG genotype of the TNF-a rs1800629 was significantly lower in patients with RA (3.9%) than in healthy controls (12.4%) (P = 0.001), and the allele rates in RA compared with controls was 2.0% versus 6.3% (P < 0.001).
Selection of candidate SNPs and sample genotyping
Subgroup and association of RA
The tag SNPs of TNF-a and TNFR1 were selected for their minor allele frequency (MAF) >10% in the Chinese population in the dbSNP database (http://www. ncbi.nlm.nih.gov/SNP/),and from results of previous published studies. Finally, we chose 3 candidate variants TNF-a (rs1800630, rs1800629) and TNFR1 (rs767455). Genomic DNA was extracted from blood samples collected in EDTA using a Relax Gene Blood DNA System (TIANGEN BIOTECH, Beijing, China) . The target polymorphisms were amplified by polymerase chain reaction (PCR) using TaKaRa Taq Hot Start Version (TaKaRa Biotechnology, Shiga, Japan) and genotyping by high resolution melting (HRM) methods using LightCycler480 (Roche, Basel, Switzerland). We randomly confirmed our genotyping results by direct sequencing by an ABI3730 sequencer. The details of the 3 SNPs can be seen in Table 1.
For a better understanding of disease aetiology, we investigated whether the observed associations were restricted to a specific subset of disease, defined by age, gender, mean disease duration, DAS28 and autoantibody status. In these subsets, no differential association could be observed for any of the 3 studied SNPs. Age was older in patients with the mutant homozygous genotype compared with heterozygous and wildtype homozygous, although this was not statistically significant. There was a trend of increasing disease duration, ESR, CRP, DAS28 and autoantibody status for some SNPs, but none were statistically significant (Tables 2 and 3).
Discussion In the present study, we have focused on polymorphisms with potential functional relevance or previ-
© 2014 John Wiley & Sons Ltd International Journal of Immunogenetics, 2014, 41, 499–502
TNF-a polymorphisms in the susceptibility to RA
501
Table 1. Genotype distributions and allele frequencies of the 3 SNPs in Chinese Han population from Shandong province Genotype Gene
SNP
Allele 1/2
TNF-a
rs1800630
C/A
rs1800629
G/A
rs767455
T/C
TNFR1
Group
11
RA Control RA Control RA Control Control
177 220 246 290 200 258 145
Allele 12
(69.1) (66.5) (96.1) (87.6) (78.1) (77.9) (43.8)
68 100 10 40 56 71 148
22 (26.6) (30.2) (3.9) (12.1) (21.9) (21.5) (44.7)
11 11 0 1 0 2 38
(4.3) (3.3) (0) (0.3) (0) ((0.6) (11.5)
P
1
0.55
422 540 502 620 456 587 438
0.001 0.46
2 (82.4) (81.6) (98.0) (93.7) (89.1) (88.7) (66.2)
90 122 10 42 56 75 224
(17.6) (18.4) (2.0) (6.3) (10.9) (11.3) (33.8)
P
OR
95% CI
1.06
0.78–1.43
0.76
3.4
1.69–6.85
C TNFRI polymorphism is associated with soluble levels and clinical activity in rheumatoid arthritis. Rheumatology International, 30, 655. You, C.G., Yin, Y.S., Xie, X.D., Ju, J., Wang, Z.P. & Chen, Y.R. (2007) Sex influences on the penetrance of IL-1beta and IL-1RN genotypes for rheumatoid arthritis in the Chinese population. Journal of International Medical Research, 35, 323.
© 2014 John Wiley & Sons Ltd International Journal of Immunogenetics, 2014, 41, 499–502
