Background: Still’s disease (SD) is a rare autoinflammatory disorder characterized by recurrent episodes of high spiking fever, transient salmon-colored skin rash, arthralgia or polyarthritis, often accompanied by neutrophilia and elevated inflammatory markers such as ferritin and transaminases. Patients may exhibit three distinct clinical patterns: monocyclic, when symptoms occur only once in a lifetime; polycyclic, when recurrent disease flares are observed; or chronic, when persistent inflammatory signs are present. The etiology of SD is not yet fully understood, it is hypothesized that environmental and genetic risk factors interact, leading to inappropriate activation of the innate immune system and subsequent overproduction of pro-inflammatory cytokines, which contribute to disease onset and progression. Among the mechanisms potentially involved in this immune dysregulation, oxidative stress may play a relevant role, as it is known to promote and amplify inflammatory responses. Excessive free radicals can damage cellular macromolecules, including proteins, lipids and DNA. The most deleterious effects are observed on mitochondrial DNA (mtDNA), which is highly susceptible to damage induced by reactive oxygen species physiologically generated during mitochondrial respiration (Xu et al., 2025). Mitochondrial DNA copy number (mtDNA-CN) is considered a biomarker of mitochondrial function (Longchamps et al., 2022), and its maintenance is regulated by several nuclear-encoded genes, including mitochondrial transcription factor A (TFAM). Today, the role of oxidative stress in SD has not been thoroughly investigated; however, its evaluation may contribute to expanding current knowledge of disease pathogenesis.

Objectives: The aim of this study was to compare mitochondrial DNA copy number (mtDNA-CN) and TFAM expression between patients with SD and healthy control subjects (CTRLs), and to determine whether SD patients exhibit increased oxidative damage to mtDNA relative to CTRLs.

Methods: Patients with SD meeting Yamaguchi criteria (Yamaguchi et al., 1992) were consecutively enrolled at the Rare Diseases Clinic of the Rheumatology Unit, AOU Policlinico Umberto I, Rome. After written informed consent, peripheral venous blood samples were collected and genomic DNA and RNA were extracted. MtDNA copy number (mtDNA-CN), oxidative damaged mtDNA, and TFAM mRNA expression levels were assessed by quantitative PCR (qPCR). For mtDNA-CN quantification, one nuclear DNA region (hemoglobin subunit beta, HGB) and one mitochondrial DNA region (NADH dehydrogenase subunit 1, ND1) were amplified, and mtDNA copy number was calculated using the formula 2 × 2^(Ct(HGB) − Ct(ND1)). To evaluate oxidative damage to mtDNA, DNA samples were treated with formamidopyrimidine DNA glycosylase (FPG), an enzyme that recognizes oxidized bases, particularly 7,8-dihydro-8-oxoguanine, and introduces strand breaks at these sites. Following enzymatic treatment, qPCR amplification of the mitochondrial ND1 gene was performed. Because DNA strand cleavage reduces qPCR amplification efficiency, treated samples displayed higher Ct values compared with untreated samples. The difference between Ct(ND1) values of treated and untreated samples (ΔCt) was used as an estimate of mtDNA oxidative damage. Statistical comparisons between SD patients and CTRLs were performed using ANOVA tests.

Results: A total of 27 consecutive patients with SD and 35 CTRLs were enrolled. SD patients showed a significantly lower mitochondrial DNA copy number (mtDNA-CN) compared with CTRLs (p = 0.006). Conversely, TFAM expression levels were significantly higher in SD patients than in CTRLs (p = 0.02). In addition, SD patients exhibited higher levels of oxidized bases in mtDNA, indicative of increased oxidative damage compared with CTRLs (p = 0.047). When patients were stratified according to disease pattern, differences were observed in both mtDNA-CN and TFAM expression levels. Specifically, patients with a chronic disease pattern showed lower mtDNA-CN and higher TFAM expression compared with those with monocyclic and polycyclic patterns. Finally, a negative correlation between mtDNA-CN and TFAM expression was observed, suggesting that increased TFAM expression may be associated with reduced mtDNA-CN (p = 0.03; r = −0.288).

Conclusions: These findings suggest that in SD, oxidative stress driven by systemic inflammation may impair mitochondrial function by damaging mtDNA and reducing mtDNA copy number. The upregulation of TFAM likely represents a compensatory mechanism to preserve mitochondrial integrity, consistent with its established role in defending mitochondria against oxidative stress. A multicenter study is ongoing to validate and expand these observations.

REFERENCES: [1] Xu X, Pang Y, Fan XM. Mitochondria in oxidative stress, inflammation and aging: from mechanisms to therapeutic advances . Signal Transduct Target Ther. 2025;10:190.

[2] Longchamps RJ, Yang SY, Castellani CA, et al. Genome-wide analysis of mitochondrial DNA copy number reveals loci implicated in nucleotide metabolism, platelet activation, and megakaryocyte proliferation . Hum Genet. 2022;141:127–146.

[3] Yamaguchi M, Ohta A, Tsunematsu T, et al. Preliminary criteria for classification of adult-onset Still’s disease . J Rheumatol. 1992;19(3):424–430.

Acknowledgments: NIL.

Disclosure of Interests: None declared.