Plasma mtDNA as a possible contributor to and biomarker of inflammation in rheumatoid arthritis

Abstract

Objectives: Neutrophil extracellular trap formation and cell-free DNA (cfDNA) contribute to the inflammation in rheumatoid arthritis (RA), but it is unknown if mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) is more abundant in the circulation. It is unclear if DNA concentration measurements may assist in clinical decision-making.

Methods: This single-center prospective observational study collected plasma from consecutive RA patients and healthy blood donors. Platelets were removed, and mtDNA and nDNA copy numbers were quantified by polymerase chain reaction (PCR).

Results: One hundred six RA patients and 85 healthy controls (HC) were recruited. Circulating median mtDNA copy numbers were increased 19.4-fold in the plasma of patients with RA (median 1.1 x10⁸ copies/mL) compared to HC (median 5.4 x10⁶ copies/mL, p<0.0001). Receiver operating characteristics (ROC) curve analysis of mtDNA copy numbers identified RA patients with high sensitivity (92.5%) and specificity (89.4%) with an area under the curve (AUC) of 0.97, p <0.0001 and a positive likelihood ratio of 8.7. Demographic, serological (rheumatoid factor (RF) positivity, anti-citrullinated protein antibodies (ACPA) positivity) and treatment factors were not associated with DNA concentrations. mtDNA plasma concentrations, however, correlated significantly with disease activity score-28- erythrocyte sedimentation rate (DAS28-ESR) and increased numerically with increasing DAS28-ESR and clinical disease activity index (CDAI) activity. MtDNA copy numbers also discriminated RA in remission (DAS28 <2.6) from HC (p<0.0001). Also, a correlation was observed between mtDNA and the ESR (p = 0.006, R= 0.29). Similar analyses showed no significance for nDNA.

Conclusion: In contrast to nDNA, mtDNA is significantly elevated in the plasma of RA patients compared with HC. Regardless of RA activity, the abundance of circulating mtDNA is a sensitive discriminator between RA patients and HC. Further validation of the diagnostic value of mtDNA testing is required.

Fig. 1

Total cfDNA (A) and mtDNA (C) in RA patients (n=106) are increased in comparison with HC individuals (n=85). nDNA (B) plasma levels however are not elevated in RA patients. Boxes represent IQR, whiskers represent the 5th and 95th percentile and dots represent outliers. The dotted horizontal line in panel C represents the optimal cut off for mtDNA testing (1.57x10⁷ copies/ml). ROC curves for total cfDNA (D), nDNA (E) and mtDNA (F) plasma concentrations to discriminate between HC (n=85) and RA patients (n=106). cfDNA, cell-free DNA; HC, healthy controls; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; RA, rheumatoid arthritis; AUC, area under the curve

Fig. 2

Circulating mtDNA copy numbers (C, F) but not total cfDNA levels (A, D) and nDNA copy numbers (B, E) are significantly elevated in RA patients compared with HC individuals throughout all strata of DAS28-ESR and CDAI activity. Furthermore, there is a trend of mtDNA to increase continuously with higher RA activity. Boxes represent IQR, whiskers represent the 5th and 95th percentile, and dots represent outliers. cfDNA, cell-free DNA; mtDNA, mitochondrial DNA; nDNA, nuclear DNA, DAS28, disease activity score-28; CDAI, clinical disease activity index; ESR, erythrocyte sedimentation rate; HC, healthy controls; R, remission; LDA, low disease activity; MDA, moderate disease activity; HDA, high disease activity