摘要
目的 分析类风湿关节炎(RA)患者外周血循环的淋巴细胞特异性蛋白酪氨酸激酶(LCK)启动子区域的甲基化特征及其与临床指标的相关性.方法 采用靶向甲基化测序方法分析比较RA与健康对照(HC)和骨关节炎(OA)患者外周血LCK启动子区域7个CpG位点的甲基化水平,并与临床信息进行相关性分析和ROC曲线构建.结果 采用非参数检验分析发现,与HC[0.53(0.50,0.57)]和OA患者[0.59(0.54,0.62)]相比,RA患者[0.63(0.59,0.68)]的整体甲基化水平升高(H=47.17,P＜0.001.同时与HC 组[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)]相比,RA 组 cg05350315_60、cg05350315_80、cg05350315_95、cg05350-315_101、cg05350315_104、cg05350315_128 和 cg05350315_142CpG 位点的甲基化水平都显著升高[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)],差异具有统计学意义(Z=-5.63,-5.89,-5.91,-5.89,-5.98,-5.95,-5.95,均 P＜0.001).与 OA 组相比[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)],RA 组 cg0535-0315_95、cg05350315_101、cg05350315_104、cg05350315_128、cg05350315_142 的 CpG 位点甲基化水平都显著升高[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)],差异具有统计学意义(Z=-3.56、-3.52、-3.60、-3.67、-3.62,P=0.036、0.042、0.031、0.030、0.030).此外,利用 Pearson相关分析发现该区域整体甲基化水平与CRP(r=0.19,P=0.004)和ESR(r=0.14,P=0.035)呈正相关.CRP(低)组[0.63(0.58,0.68)]的LCK启动子区域整体甲基化水平高于CRP(高)组[0.65(0.61,0.70)],差异有统计学意义(Z=2.60,P=0.009).最后,通过构建ROC曲线,验证外周血LCK启动子区域甲基化水平作为RA患者,特别是血清阴性RA患者与HC、OA组的鉴别效能,曲线下面积(AUC)值(95％CI)为0.78(0.63,0.93).结论 本研究提供了 RA患者外周血LCK启动子区域甲基化状态和甲基化单倍型模式特征,该区域整体甲基化水平与炎症水平呈正相关,同时可用于鉴别血清阴性RA患者与HC、OA患者.
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.
基金项目
国家自然科学基金(82074234)
华东片区中西医结合关节病专科联盟项目(2021-2023)()
上海市何东仪名医工作室建设项目(SHGZS-202220)
NETL
NSTL
万方数据