ABSTRACT
Objective:To analyze the methylation characteristics of the lymphocyte-specific protein-tyrosine kinase (LCK) promoter region in the peripheral blood circulation of rheumatoid arthritis (RA) patients and its correlation with clinical indicators.Methods:Targeted methylation sequencing was used to compare the methylation levels of 7 CpG sites in the LCK promoter region in the peripheral blood of RA patients with healthy controls (HC) and osteoarthritis (OA) patients. Correlation analysis and ROC curve construction were performed with clinical information.Results:Non-parametric tests revealed that compared with HC [0.53(0.50, 0.57)] and OA patients [0.59(0.54, 0.62), H=47.17, P<0.001], RA patients [0.63(0.59, 0.68)] exhibited an overall increase in methylation levels. Simultaneously, when compared with the HC group [0.38(0.35, 0.41), 0.59(0.55, 0.63), 0.60(0.55, 0.64), 0.59(0.55, 0.63), 0.58(0.53, 0.62), 0.45(0.43, 0.49), 0.57(0.54, 0.61)], the RA group [0.46(0.42, 0.49), 0.70(0.65, 0.75), 0.70(0.66, 0.76), 0.70(0.65, 0.75), 0.69(0.64, 0.74), 0.55(0.51, 0.59), 0.68(0.63, 0.73)] showed a significant elevation in methylation levels at CpG sites cg05350315_60, cg05350315_80, cg05350315_95, cg05350315_101, cg05350315_104, cg05350315_128, and cg05350315_142, with statistically significant differences ( Z=-5.63, -5.89, -5.91, -5.89, -5.98, -5.95, -5.95, all P<0.001). Compared with the OA group [0.65(0.59, 0.69), 0.65(0.60, 0.69), 0.64(0.58, 0.68), 0.50(0.45, 0.54), 0.63(0.58, 0.67)], the RA group [0.70(0.66, 0.76), 0.70(0.65, 0.75), 0.69(0.64, 0.74), 0.55(0.51, 0.59), 0.68(0.63, 0.73)] exhibited a significant increase in methylation levels at CpG sites cg05350315_95, cg05350315_101, cg05350315_104, cg05350315_128, and cg05350315_142, with statistically significant differences ( Z=-3.56, -3.52, -3.60, -3.67, -3.62; P=0.036, 0.042, 0.031, 0.030, 0.030). Furthermore, Pearson correlation coefficient analysis revealed a positive correlation between the overall methylation level in this region and C-reactive protein (CRP) ( r=0.19, P=0.004) and erythrocyte sedimentation rate ( r=0.14, P=0.035). The overall methylation level of the LCK promoter region in the CRP (low) group [0.63 (0.58, 0.68)] was higher than that in the CRP (high) group [0.65(0.61, 0.70)], with statistically significant differences ( Z=2.60, P=0.009). Finally, by constru-cting a ROC curve, the discriminatory efficacy of peripheral blood LCK promoter region methylation levels for identifying RA patients, especially seronegative RA patients, from HC and OA groups was validated, with an AUC value of 0.78 (95% CI: 0.63, 0.93). Conclusion:This study provides insights into the methylation status and methylation haplotype patterns of the LCK promoter region in the peripheral blood of RA patients. The overall methylation level in this region is positively correlated with the level of inflammation and can be used to differentiate seronegative RA patients from the HC and OA patients.
ABSTRACT
Objective:In general, patients with seropositive rheumatoid arthritis (RA) are considered to show an aggressive disease course. However, the relationship between the two subgroups in disease severity is controversial. Our study is aimed to compare the clinical characteristics and prognosis of double-seropositive and seronegative RA in China through a real-world large scale study.Methods:RA patients who met the 1987 American College of Rheumatology (ACR) classification criteria or the 2010 ACR/European Anti-Rheumatism Alliance RA classification criteria, and who attended the 10 hospitals across the country from September 2015 to January 2020, were enrolled. According to the serological status, patients were divided into 4 subgroups [rheumatoid factor (RF)(-) anti-cyclic citrullinated peptide (CCP) antibody (-), RF(+), RF(+) anti-CCP antibody(+), anti-CCP antibody(+)] and compared the disease characteristics and treatment response. One-way analysis of variance was used for measurement data that conformed to normal distribution, Kruskal-Wallis H test was used for measurement data that did not conform to normal distribution; paired t test was used for comparison before and after treatment within the group if the data was normally distributed else paired rank sum test was used; χ2 test was used for count data. Results:① A total of 2 461 patients were included, including 1 813 RF(+) anti-CCP antibody(+) patients (73.67%), 129 RF(+) patients (5.24%), 245 RF(-) anti-CCP antibody(-) patients (9.96%), 74 anti-CCP antibody(+) patients (11.13%). ② Regardless of the CCP status, RF(+) patients had an early age of onset [RF(-) anti-CCP antibody(-) (51±14) years old, anti-CCP antibody(+) (50±15) years old, RF(+) anti-CCP antibody(+) (48±14) years old, RF(+)(48±13) years old, F=3.003, P=0.029], longer disease duration [RF(-) anti-CCP antibody(-) 50 (20, 126) months, anti-CCP antibody(+) 60(24, 150) months, RF(+) anti-CCP antibody(+) 89(35, 179) months, RF(+) 83(25, 160) months, H=22.001, P<0.01], more joint swelling counts (SJC) [RF(-) anti-CCP antibody(-) 2(0, 6), Anti-CCP antibody(+) 2(0, 5), RF(+) anti-CCP antibody(+) 2(0, 7), RF(+) 2(0, 6), H=8.939, P=0.03] and tender joint counts (TJC) [RF(-) anti-CCP antibody(-) 3(0, 8), anti-CCP antibody(+) 2(0, 6), RF(+) anti-CCP antibody(+) 3(1, 9), RF(+) 2(0, 8), H=11.341, P=0.01] and the morning stiff time was longer [RF(-) anti-CCP antibody(-) 30(0, 60) min, anti-CCP antibody(+) 20(0, 60) min, RF(+) anti-CCP antibody(+) 30(10, 60) min, RF(+) 30(10, 60) min, H=13.32, P<0.01]; ESR [RF(-) anti-CCP antibody(-) 17(9, 38) mm/1 h, anti-CCP antibody(+) 20(10, 35) mm/1 h, RF(+) anti-CCP antibody(+) 26(14, 45) mm/1 h, RF(+) 28(14, 50) mm/1 h, H=37.084, P<0.01] and CRP [RF(-) anti-CCP antibody(-) 2.3 (0.8, 15.9) mm/L, Anti-CCP antibody(+) 2.7(0.7, 12.1) mm/L, RF(+) anti-CCP antibody(+) 5.2(1.3, 17.2) mm/L, RF (+) 5.2(0.9, 16.2) mm/L, H=22.141, P<0.01] of the RF(+)patients were significantly higher than RF(-) patients, and RF(+) patients had higher disease severity(DAS28-ESR) [RF(-) anti-CCP antibody(-) (4.0±1.8), anti-CCP antibody(+) (3.8±1.6), RF(+) anti-CCP antibody(+) (4.3±1.8), RF(+) (4.1±1.7), F=7.269, P<0.01]. ③ The RF(+) anti-CCP antibody(+) patients were divided into 4 subgroups, and it was found that RF-H anti-CCP antibody-L patients had higher disease severity [RF-H anti-CCP antibody-H 4.3(2.9, 5.6), RF-L anti-CCP antibody-L 4.5(3.0, 5.7), RF-H anti-CCP antibody-L 4.9(3.1, 6.2), RF-L anti-CCP antibody-H 2.8(1.8, 3.9), H=20.374, P<0.01]. ④ After 3-month follow up, the clinical characteristics of the four groups were improved, but there was no significant difference in the improvement of the four groups, indicating that the RF and anti-CCP antibody status did not affect the remission within 3 months. Conclusion:Among RA patients, the disease activity of RA patients is closely related to RF and the RF(+) patients have more severe disease than RF(-) patients. Patients with higher RF titer also have more severe disease than that of patients with low RF titer. After 3 months of medication treatment, the antibody status does not affect the disease remission rate.