Wen-Chan Tsai

Global DNA methylation, DNMT1, and MBD2 in patients with rheumatoid arthritis.

OBJECTIVES To investigate the associations of DNA methylation levels and mRNA expressions of DNA cytosine-5-methyltransferase 1 (DNMT1) and methyl CpG-binding domain 2 (MBD2) with rheumatoid arthritis (RA). METHODS The global methylation status of DNA was measured in 65 patients with RA and 64 healthy controls by the ELISA method. DNMT1 and MBD2 mRNA were also detected in 177 RA patients and 95 controls using the quantitative real-time polymerase chain reaction method. RESULTS The global methylation of DNA was significantly decreased in the RA patients compared to the controls (p=0.005, 95% CI=0.0835-0.4503). The patients with RA had higher expressions of DNMT1 and MBD2 mRNA than the controls (p<0.001, 95% CI=-0.0024 to -0.0053 and p<0.001, 95% CI=-0.0079 to -0.0167, respectively). We also found that the MBD2 mRNA level was not related to the disease activity of RA. However, the expression of DNMT1 mRNA tended to be associated with the disease activity of RA (p=0.08). The levels of DNA methylation and DNMT1 mRNA were significantly decreased in the patients with anti-CCP antibody compared with those without (p=0.005, 95% CI=-0.7333 to -0.1373 and p=0.003, 95% CI=-0.0071 to -0.0022, respectively). The differences in the methylation level and expressions of DNMT1 and MBD2 were not significant between the patients treated with and without anti-TNFα biological agents (Enbrel or Humira). CONCLUSION This study demonstrated that the RA patients have a significantly lower level of DNA methylation than the controls. Moreover, RA patients have higher expressions of DNMT1 and MBD2 mRNA. The anti-TNFα biological agents do not seem to affect DNA methylation and mRNA expressions of DNMT1 and MBD2 in RA patients.

Programmed Death-1 Gene Polymorphisms in Patients With Systemic Lupus Erythematosus in Taiwan

To investigate the role of programmed cell death-1 (PD-1) gene polymorphisms in the development of systemic lupus erythematosus (SLE) in Taiwan, 109 patients with SLE and 100 healthy controls were enrolled in this study. The PD-1 gene polymorphisms were determined by the method of polymerase chain reaction/restriction fragment length polymorphism. This study showed that the genotype distributions of PD-1 7209 C/T polymorphisms were significantly different between the patients with SLE and controls (P=0.002, Pc=0.018). The frequencies of the PD-1 7209 C/C genotype and PD-1 7209 C allele were significantly higher in the patients with SLE than those of the controls (P=0.001, OR=2.6, 95% CI=1.5–4.6, and P=0.002, OR=2.1, 95% CI=1.3–3.4, Pc=0.018, respectively). Moreover, the association of PD-1 7209 C with susceptibility to SLE was independent of the PD-1 ligand. This study also showed that the PD-1-536 A 7146 G 7209 C 7499 G haplotype was associated with the development of SLE in Taiwan.

Detecting Epstein-Barr virus DNA from peripheral blood mononuclear cells in adult patients with systemic lupus erythematosus in Taiwan

Epstein-Barr virus (EBV) has been found by many serology studies to be associated with systemic lupus erythematosus (SLE). However, the results of DNA studies have been conflicting. Therefore, instead of antibody to EBV, we studied the association between EBV DNA and SLE. In this case-control study in Taiwan, we enrolled 87 SLE patients and 174 age- and sex-matched controls. Peripheral blood mononuclear cells of SLE patients and matched controls were tested for EBV DNA by polymerase chain reaction (PCR) and Southern blot. Of the 87 SLE patients, 71 (81.6%) were found to be positive for EBV DNA, while 85 (48.9%) of the 174 controls (odds ratio 4.64, 95% confidence interval 2.50–8.62, P<0.0001) were positive. While the EBV DNA-positive rate did not decline with age in SLE patients (P>0.05), it did decline with age in controls (P<0.05). Furthermore, based on a real-time quantitative PCR study, we have found a significant difference between EBV viral load in SLE and controls (P=0.008). Therefore, in our molecular study of DNA level, we found evidence for the association of EBV infection and SLE, suggesting that EBV contributes, if not to the development of SLE, then to disease perpetuation.

Global DNA methylation, DNMT1, and MBD2 in patients with systemic lupus erythematosus

To investigate the associations of DNA methylation levels and mRNA expressions of DNA cytosine-5-methyltransferase 1 (DNMT1) and methyl CpG-binding domain 2 (MBD2) with systemic lupus erythematosus (SLE), 108 patients with SLE and 97 healthy controls were enrolled in this study. DNA and total RNA were extracted from the peripheral blood mononuclear cells of the SLE patients and the controls. The global methylation levels of DNA were measured in 63 patients with SLE and 68 healthy controls by the ELISA method. DNMT1 and MBD2 mRNA were also detected in 108 SLE patients and 97 controls using the quantitative real-time polymerase chain reaction method. The global methylation level of DNA was significantly decreased in the SLE patients in comparison with that in the controls (p < 0.001, 95% CI = 0.1573–0.5052). The patients with SLE have higher expressions of DNMT1 and MBD2 mRNA than the controls (p < 0.001, 95% CI = −0.0049 – −0.0019 and p = 0.001, 95% CI = −0.0119 – −0.0029, respectively). We also found that there were no significant differences in the methylation level and the expression of DNMT1 and MBD2 mRNA between the active and the inactive SLE patients. A positive correlation was also found between DNMT1 and MBD2 mRNA expressions in the SLE patients (p < 0.001). This study demonstrated that the patients with SLE had a significantly lower level of DNA methylation than the controls. The expression of both DNMT1 and MBD2 mRNA was significantly increased in the SLE patients compared with the controls. This study also showed a positive correlation between DNMT1 and MBD2 mRNA levels in the patients with SLE.

The TNF2 allele does not contribute towards susceptibility to systemic lupus erythematosus.

The uncommon allele (TNF2) of a polymorphism in the promoter region of the tumor necrosis factor-alpha (TNF-alpha) gene has been reported to be increased in Caucasian systemic lupus erythematosus (SLE) patients (associated with HLA-DR3). To investigate whether TNF2 contributes towards susceptibility to Chinese SLE patients (not associated with HLA-DR3), 100 patients with SLE and 107 controls were studied. The frequency of TNF2 allele in controls was 0.140. There was a strong association between TNF2 allele and HLA-DR3 (P < 10(-8)) in controls. The frequency of TNF2 allele in SLE patients was 0.15. There was no difference in frequencies of TNF2 allele between patients and controls. This finding strongly suggests that TNF2 does not play a direct role in the susceptibility of SLE.

Suppressor of cytokine signaling 1 gene expression and polymorphisms in systemic lupus erythematosus

With the aim of investigating the role of suppressor of cytokine signaling 1 (SOCS1) in the pathogenesis of systemic lupus erythematosus, 107 patients with systemic lupus erythematosus, 101 healthy controls, and 151 patients with ankylosing spondylitis were enrolled in this study. SOCS1 mRNA level was measured by the method of quantitative real-time polymerase chain reaction. SOCS1 polymorphisms were detected by the polymerase chain reaction/restriction fragment length polymorphisms method. Systemic lupus erythematosus disease activity was evaluated with the SLEDAI. This study showed that the SOCS1 mRNA expression was significantly higher in the patients with systemic lupus erythematosus than in the healthy controls (p = 0.0014). Patients with active systemic lupus erythematosus had a higher expression of SOCS1 mRNA than the patients with inactive systemic lupus erythematosus (p = 0.035). There was no significant difference in the frequencies of the SOCS1-1478CA/del polymorphisms among the patients with systemic lupus erythematosus, healthy controls, and patients with ankylosing spondylitis. The genotype frequency of the SOCS1-1478 polymorphisms in the dominant model (CA/del+del/del versus CA/CA) was significantly decreased in the patients with thrombocytopenia compared with those without thrombocytopenia (pc = 0.035). Moreover, the allele frequency of SOCS1-1478del was also significantly lower in the patients with thrombocytopenia than in those without thrombocytopenia (p c = 0.02). In conclusion, this study demonstrated that the expression of SOCS1 mRNA was significantly increased in patients with systemic lupus erythematosus. Moreover, SOCS1 mRNA levels in patients with active systemic lupus erythematosus were significantly higher than those in the inactive patients. We also found that the systemic lupus erythematosus patients with thrombocytopenia have a lower frequency of SOCS1-1478del compared with patients without thrombocytopenia. Lupus (2010) 19, 696—702.

IkBα Promoter Polymorphisms in Patients with Primary Sjögren’s Syndrome

IntroductionTo investigate the association of IkBα promoter polymorphisms with the development of primary Sjögren’s syndrome in Taiwan, 98 patients with primary Sjögren’s syndrome and 110 unrelated healthy controls were enrolled in this study.Materials and MethodsThe IκBα −881 A/G, IκBα −826 C/T, IκBα −550 A/T, IκBα −519 C/T, and IκBα −297 C/T polymorphisms were determined by the methods of polymerase chain reaction/restriction fragment length polymorphism.ResultsThis study demonstrated that the genotype frequencies of IκBα −826 C/T and IκBα −826 T/T, in comparison with that of IκBα −826 C/C, were significantly higher in the patients with primary Sjögren’s syndrome than in the controls. The allele frequency of IκBα −881 G was significantly decreased in the patients with primary Sjögren’s syndrome compared with that of the controls. In contrast, the allele frequency of IκBα −826 T was significantly higher in the patients with primary Sjögren’s syndrome than in the controls. The similar findings could also be found in the allele carriage frequencies. The patients with primary Sjögren’s syndrome had lower allele carriage frequencies of IκBα −881 G and IκBα −826 C, and a higher allele carriage frequency of IκBα −826 T. We also found that the estimated haplotype frequency of IκBα −881A-826T-550A-519C-297C was significantly increased in the patients with primary Sjögren’s syndrome in comparison with that of the controls.DiscussionThis study demonstrated that the IkBα −826T allele and IkBα −881A-826T-550A-519C-297C haplotype were associated with susceptibility to primary Sjögren’s syndrome in Taiwan. However, these findings may not be disease-specific but may be related to inflammatory responses.

IκBα Promoter Polymorphisms in Patients with Systemic Lupus Erythematosus

To investigate the associations of IκBα gene polymorphisms with the development and clinical manifestations of systemic lupus erythematosus (SLE), 110 patients with SLE and 120 unrelated healthy controls were enrolled in this study. The IκBα −881 A/G, −826 C/T, −550 A/T, −519 C/T, and −297 C/T polymorphisms were determined by the polymerase chain reaction/reaction fragment length polymorphism method. The genotype frequency of IκBα −826 C/T in the patients with SLE was significantly higher than that of the controls (p = 0.003, OR = 2.2, 95% CI = 1.3–3.9). The SLE patients also have significantly higher carriage rate of IκBα −826 T than the controls (p = 0.01, OR = 2.0, 95% CI = 1.2–3.4). We also found that the estimated haplotype frequency of IκBα −881A −826T −550A −519C −297C was significantly increased in the patients with SLE in comparison with that of the controls. This study also demonstrated that the association of IκBα −826 T with SLE was independent of HLA-DR15, which is associated with susceptibility to SLE in Taiwan. Moreover, a synergistic effect could also be found between IκBα −826 T and HLA-DR15. IκBα −826 T is associated with the development of SLE in Taiwan. The IκBα −881A −826T −550A −519C −297C haplotype is also associated with susceptibility to SLE. This study also demonstrated that IκBα -881G was associated with the occurrence of vasculitis in SLE patients. IκBα −550T might be a protective factor for the development of malar rash.

Demethylation within the proximal promoter region of human estrogen receptor alpha gene correlates with its enhanced expression: Implications for female bias in lupus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease primarily affecting women. Previous studies have indicated that sex hormone estrogens contribute to the female predilection of SLE. Estrogen regulates gene expression by translocating estrogen receptors (ER) α and β into the nucleus where they induce transcription by binding to estrogen response elements of target genes. We have previously observed that expression of ERα gene and protein in lupus patients is significantly higher than in healthy controls and that estradiol up-regulates calcineurin expression via over-expression of ERα gene in SLE. However, the pathogenesis of over-expression of ERα gene is unknown. Here we report that enhanced expression of ERα mRNA and protein in SLE and rheumatoid arthritis is associated with DNA demethylation within the proximal promoter region located between -232 and +81 base pair relative to transcription start site of human ERα gene (GenBank Accession no. AL356311.6). The frequency of DNA demethylation was comparable between male and female. These findings suggest that estrogen and demethylated ERα promoter associated up-regulated ERα genes are two critical factors in the gender biased development of autoimmune diseases besides genetic factor.

Long-Term Frequent Use of Non-Steroidal Anti-Inflammatory Drugs Might Protect Patients with Ankylosing Spondylitis from Cardiovascular Diseases: A Nationwide Case-Control Study

The objective of this case-control study was to investigate the risk of cardiovascular disease (CVD) following non-steroidal anti-inflammatory drug (NSAID) use in patients with ankylosing spondylitis (AS). A total of 10,763 new AS patients were identified from the National Taiwan Health Insurance claims database during the period from 1997 to 2008. In all, 421 AS patients with CVD were recruited as cases, and up to 2-fold as many sex- and age-matched controls were selected. Logistic regression models were used to estimate the odds ratio (OR) between NSAID use and CVD incidence. The medication possession rate (MPR) was used to evaluate NSAID exposure during the study period. AS patients had increased risk of CVD (OR, 1.68; 95% confidence interval (CI), 1.57 to 1.80). Among frequent (MPR≥80%) COX II users, the risks for all types of CVD were ten times lower than those among non-users at 24 months (OR, 0.08; 95% CI, 0.01 to 0.92). Among frequent NSAID users, the risks of major adverse cardiac event (MACE) were significantly lower at 12 months (OR, 0.23; 95% CI, 0.07 to 0.76)—a trend showing that longer exposure correlated with lower risk. Regarding non-frequent NSAID users (MPR<80%), short-term exposure did carry higher risk (for 6 months: OR, 1.41; 95% CI, 1.07 to 1.86), but after 12 months, the risk no longer existed. We conclude that long-term frequent use of NSAIDs might protect AS patients from CVD; however, NSAIDs still carried higher short-term risk in the non-frequent users.