| Objectives: Systemic lupus erythematosus(SLE)is the most important disabling and fatal autoimmune disease at present.Kidney is the most frequently involved organ of SLE,and lupus nephritis(LN)is an important cause of death in patients with SLE.It is of great significance for the diagnosis and treatment of SLE to quickly and accurately determine the disease activity and renal injury in patients with SLE.More and more evidences have shown that plasma circulating cell-free DNA(cfDNA)concentration in patients with SLE is significantly higher than that in healthy people,and its level is related to disease activity and renal injury.The use of cfDNA as a new biomarker for SLE has become the focus of many studies.However,there is currently no effective standard for the clinical application of cfDNA.In this study,the correlation between plasma cfDNA and clinical characteristics in SLE patients was analyzed,and combined models of cfDNA and clinical indicators were established to explore the diagnostic value of cfDNA in disease activity and renal injury in SLE patients.Methods: Blood plasma samples were collected from 127 patients with SLE,and the conditions and laboratory results were recorded.Plasma cfDNA was extracted by Mag MAX Cell-Free DNA Isolation Kit,and the concentration of DNA after extraction was determined by Qubit ds DNA HS Assay Kit.The size and distribution of DNA fragments in the samples were measured by Agilent 2100 Bioanalyzer,and the fragmentation index of cfDNA was calculated.This study analyzed the correlation between the concentration and fragmentation index of cfDNA and different clinical indicators.This study also explored the clinical diagnostic value of the combined diagnosis model established by combining cfDNA with clinical information in judging the degree of SLE disease activity and determining renal injury.Results:1.The concentration of cfDNA was positively correlated with the fragmentation index(P<0.0001,r=0.6007).2.The concentration of cfDNA in the patients with positive anti-U1 RNP antibody(0.847±1.171 ng/ul vs.0.602±1.305 ng/ul,P=0.012)or positive anti-SSA antibody(0.923±1.527 ng/ul vs.0.514±0.732 ng/ul,P=0.024)was higher than that in the patients with negative antibody.The concentration of cfDNA in those with positive anti-ds DNA antibodies(0.639±1.332 ng/ul vs.0.803±1.165 ng/ul,P=0.021)or positive anti-nucleosome antibodies(0.935±1.654 ng/ul vs.1.235±1.463 ng/ul,P=0.039)was lower than that in those with negative antibodies.There was no significant difference in fragmentation index between positive and negative antibody groups(P>0.05).3.The concentration of cfDNA in active patients was significantly higher than that in inactive patients(0.901±1.439 ng/ul vs.0.354±0.339 ng/ul,P=0.043),and the same results were observed in the range 153-198bp(856.93±945.3 pg/ul vs.329.45±274.39 pg/ul,P=0.005)and 300-699bp(173.04±194.36 pg/ul vs.63.7±63.43 pg/ul,P=0.045).There was no difference in the fragmentation index between the two groups(P=0.213),and there was no significant difference in the proportion of patients with high cfDNA fragmentation index between the two groups(51.1% vs.64.7%,P=0.333).There was a negative correlation between cfDNA concentration and the titer of anti-ds DNA antibody(P=0.0337,r=-0.3009),but no correlation between cfDNA concentration and other clinical indicators.There was no correlation between cfDNA fragmentation index and clinical indicators(P>0.05).There was no significant difference in clinical indicators between patients with high cfDNA fragmentation index and patients with low cfDNA fragmentation index(P>0.05).4.Active Index,a combined model established by cfDNA concentration,complement C3,neutrophils percentage,erythrocyte sedimentation rate,and albumin,was effective in determining whether patients with SLE were in the Active stage of disease.The AUROC was 0.9001(95% confidence interval 0.8131-0.9871),and the best cut-off value was 0.7765,with a sensitivity of 76.5% and specificity of 89.6%(P<0.0001).Active index was negatively correlated with SLEDAI score(P<0.0001,r=-0.7031).5.The concentration of cfDNA in lupus nephritis patients was significantly lower than that in non-lupus nephritis(NLN)patients(0.632±0.896 ng/ul,n=53 vs.1.309±1.945ng/ul,n=35,P=0.015),and there was no significant difference in fragmentation index between the two groups.6.In patients with lupus nephritis,cfDNA concentration was positively correlated with neutrophils count(P=0.0417,r=0.4021)and negatively correlated with anti-ds DNA antibody titers(P=0.0296,r=-0.4189).Both neutrophils count and e GFR were the main factors affecting the level of cfDNA in patients with LN.Neutrophils count had a positive effect on cfDNA concentration(β=0.601,P=0.002),while e GFR(β=-0.378,P=0.041)had a negative effect on cfDNA concentration.7.The combination model LN index,which was established using cfDNA concentration,Ig G level,and serum creatinine level,had a good diagnostic efficacy for renal injury in patients with active SLE.The AUROC was 0.8235(95% confidence interval 0.6796-0.9675),and the best cut-off was-23.7367.The sensitivity was 76.5%,and the specificity was 78.6%(P=0.0022).LN index was positively correlated with 24-hour urinary protein quantitation(P=0.0039,r=0.5661),urinary RBC count(P=0.0166,r=0.4269)and serum creatinine level(P=0.0005,r=0.5915).Conclusions: These results suggested that the concentrations of cfDNA are different in SLE patients with different disease states.The concentration of cfDNA was higher in active SLE than in inactive SLE,and higher in NLN patients than in LN patients.The diagnostic model established by cfDNA concentration combined with other indicators had good diagnostic efficacy for disease activity and kidney injury of SLE,and could reflect the degree of disease activity and kidney injury.By collecting the peripheral blood to obtain the concentration of cfDNA,not only less trauma,can be collected at any time,but also real-time and dynamic monitoring of the changes of the disease.Using cfDNA as a new biomarker of SLE and LN has potential clinical value. |
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