Discussion

The Middle East, Mediterranean and the Far East regions had higher prevalence of Behçet’s disease, typically emerging in the third decade of life. As a chronic inflammatory disease, its exact cause remains unclear, but genetic predisposition, immune dysfunction, and environmental factors are key contributors17–19.

CircRNAs produced in human cells are key elements of the powerful molecular network regulating gene expression. CircRNAs are produced from many chromosomes, including chromosomes 1,2,3, and 6, where these chromosomes regulate immune and inflammatory pathways through mechanisms like microRNA sponging and gene modulation6,20–22.

No existing research, to our knowledge, has reported on circRNA-FUNDC1 in Behçet’s disease. However, given the established role of circRNAs in immune system regulation and inflammatory diseases, circRNA-FUNDC1 may contribute to Behçet’s disease pathogenesis23.

TNF-alpha contributes significantly to the inflammatory cascade of Behçet’s disease, driving immune system hyperactivity and tissue damage. It promotes neutrophil activation, increases vascular permeability, and enhances the release of other inflammatory mediators, contributing to BD’s hallmark symptoms, such as oral ulcers, uveitis, and vasculitis24,25.

In this study, we evaluated serum of circRNA-FUNDC1 expression levels and the concentration level of TNF-α of Behçet patients as well as healthy individuals. Results revealed that both biomarkers were upregulated in the Behçet’s patient group compared to controls.

Currently, there is limited direct research linking circRNA-FUNDC1 to BD. However, its host gene (FUNDC1) plays a pivotal function in mitophagy regulation, which is crucial for cellular homeostasis and has implications in various diseases. A review by Li et al. (2023) discusses the involvement of 31 different circRNAs in processes like autophagy, apoptosis, inflammation and oxidative stress following (IS) ischemic stroke. This highlights the potential of circRNAs as therapeutic targets and biomarkers in inflammatory conditions7,26.

In addition, circRNA-FUNDC1 has been previously studied in ischemic stroke, where it has been shown to regulate endothelial cell injury, indicating that knockdown of circRNA-FUNDC1 alleviates oxygen-glucose deprivation-induced damage in microvascular endothelial cells of the brain by targeting the microRNA-375/PTEN axis. This suggests that circRNA-FUNDC1 may contribute to inflammatory responses in ischemic stroke by modulating key molecular pathways23,27.

TNF-α is a well-established pro-inflammatory cytokine that plays a central role in the immunopathogenesis of Behçet’s disease (BD), contributing to immune dysregulation and persistent inflammation. Increased TNF-α production by CD4 + and CD8 + T cells, macrophages, and natural killer cells, together with elevated serum TNF-α levels in patients with BD compared with healthy controls, has been consistently reported, supporting its role in disease activity and maintenance14,28,29.

Similar elevations have also been observed in other autoimmune inflammatory diseases, such as juvenile chronic arthritis (JCA), further highlighting the broader pathogenic role of TNF-α in chronic inflammatory disorders30.

In addition, when measuring the serum biomarkers concerning the disease severity, it was detected that circRNA-FUNDC1 and TNF-α levels tended to elevate with the severity of the disease activity, where the BDCAF score gets higher in active behcet patients than inactive patients.

In agreement with our findings, a study involving 30 Egyptian Behçet’s patients and 20 healthy subjects, researchers found that TNF-α levels in serum were higher in patients, particularly those with active disease28.

Several studies have demonstrated that the BDCAF scores were higher in active patients when compared to inactive patients. A study involving 74 BD patients found that 16.2% were classified as active based on BDCAF scores, while 83.8% were inactive. BDCAF was higher in active patients than inactive individuals, concluding it to be a crucial factor contributing to functional disability in BD patients31.

Results also revealed a significant variation between circRNA-FUNDC1 and patients with negative musculoskeletal and CNS manifestations, with higher expression levels of circRNA-FUNDC1 than in positive patients. Although this finding appears counterintuitive, it may reflect the heterogeneous clinical nature of Behçet’s disease and the possibility that circRNA-FUNDC1 is differentially regulated among distinct disease phenotypes rather than being uniformly associated with all manifestations. Additionally, the relatively small number of patients with musculoskeletal and CNS involvement may have influenced these subgroup analyses. Another possible explanation is that circRNA-FUNDC1 may exert a compensatory or protective response in certain clinical settings. Therefore, these findings should be interpreted cautiously and warrant further investigation in larger, multicenter studies. In addition, the level of circRNA-FUNDC1 tended to be significantly higher in patients who received Azathioprine as an immunosuppressant than in those who did not. MeanWhile patients who did not take Cyclosporin had higher levels of circRNA-FUNDC1 than those with negative intake of Cyclosporin, with a slightly significant difference between groups. Our results also revealed that there was a significantly higher level of circRNA-FUNDC1 in patients who received Steroids than in those who did not.

A substantial proportion of patients received combination immunosuppressive therapy, and some medication categories contained relatively few patients in either the treatment or non-treatment groups resulting in unstable regression estimates and increase the likelihood of multicollinearity. Consequently, the independent effects of individual medications on biomarker expression could not be completely disentangled. Therefore, the findings from the medication-adjusted models should be interpreted cautiously and require confirmation in larger independent cohorts.

Our findings showed that for the concentration level of TNF-α, patients with oral ulcers had higher levels of TNF-α than those who did not, with a significant difference between both groups. As for organ manifestations, a significant difference was detected as regards patients with skin manifestations involvement that those who had negative skin issues. Also, TNF-α concentration levels tended to be higher in patients who received Hydroxychloroquine as an immunosuppressant.

A study analyzing 60 BD patients showed that those with active oral ulcers had significantly higher TNF-α levels compared to those without ulcers. These findings highlight TNF-α as a key inflammatory marker in BD, suggesting its potential role as a biomarker for the activity of the disease32.

Another study examined 45 BD patients reported significantly elevated TNF-α levels in the gingival crevicular fluid of those with recurrent oral ulcers. The results support TNF-α involvement in BD-related mucosal inflammation, reinforcing its potential as a therapeutic target33.

In agreement with our findings, a study involving 37 BD patients, of whom 19 had active oral ulcers, found that those with ulcers exhibited significantly higher salivary levels of TNF-α compared to patients without ulcers and healthy controls. This suggests that elevated salivary TNF-α is associated with oral ulcer activity in BD34.

The observed associations between circRNA-FUNDC1 expression and clinical features of Behçet’s disease suggest a relationship with overall disease activity rather than specific organ involvement. In the present study, higher circRNA-FUNDC1 levels were observed in patients with active disease as reflected by higher BDCAF scores, supporting its potential role as a marker of systemic inflammatory burden in Behçet’s disease17,35. However, the variability observed across different clinical manifestations, including musculoskeletal and CNS involvement, indicates that circRNA-FUNDC1 is unlikely to serve as an organ-specific biomarker and may instead reflect heterogeneous immune–inflammatory and mitochondrial stress responses across Behçet’s disease phenotypes. The association with immunosuppressive therapy likely reflects confounding by indication, as patients receiving treatment typically represent more severe disease. Overall, these findings suggest that circRNA-FUNDC1 may act as a biomarker of disease activity and systemic inflammation in Behçet’s disease rather than a determinant of specific clinical manifestations.

Several associations reached nominal statistical significance in the unadjusted analyses however, none remained significant after correction for multiple testing, suggesting that these findings should be interpreted cautiously and require confirmation in larger independent cohorts.

Although a direct regulatory relationship between circRNA-FUNDC1 and TNF-α has not yet been established, there is a biologically plausible indirect connection through FUNDC1-mediated mitophagy and inflammatory signaling. FUNDC1 is a key receptor involved in mitophagy and mitochondrial quality control. Impaired mitophagy results in the accumulation of damaged mitochondria, excessive production of reactive oxygen species (ROS), and the release of mitochondrial damage-associated molecular patterns, which subsequently activate inflammatory pathways and promote the production of pro-inflammatory cytokines, including TNF-α36,37.

Moreover, several studies have highlighted the role of FUNDC1 in inflammatory diseases and demonstrated that FUNDC1-mediated mitophagy participates in the regulation of immune and inflammatory responses37,38. Since TNF-α is a central cytokine in the pathogenesis of Behçet’s disease and a major therapeutic target, it was included in the present study as a representative inflammatory marker to explore whether circRNA-FUNDC1 expression is associated with the inflammatory status of Behçet’s disease.

However, no significant correlation was observed between circRNA-FUNDC1 and TNF-α levels in our cohort. This finding suggests that circRNA-FUNDC1 may reflect a pathogenic mechanism in Behçet’s disease that is independent of the classical TNF-α-mediated inflammatory pathway. Therefore, the analysis of TNF-α should be considered exploratory, and no direct mechanistic interaction or causal relationship can be inferred from the present cross-sectional study37,38.

Although demographic data (age and sex) and selected clinical information, including family history and associated diseases/comorbidities, were collected for the healthy control group, other clinical variables were not systematically collected or controlled for. Therefore, the findings observed for circRNA-FUNDC1 ROC curve should be interpreted with caution, and future studies should include more comprehensive characterization of healthy controls and appropriate disease control groups to further validate these results.Therefore, further studies involving larger, multicenter populations and external validation are warranted to confirm the diagnostic utility and generalizability of circRNA-FUNDC1 as a biomarker for Behçet’s disease.