Subjects and methods

Fifty participants meeting the criteria outlined in 1975 Declaration of Helsinki were included in this study, with an allowable absolute error/precision margin of 1.37% and a confidence level (CI) of 95% with total sample size is 49.23. Agreeing on a total of 100 subjects subdivided equally into two groups with matched age and sex, patients with Behçet’s disease and controls to be in the study. The study approval was obtained from the Faculty of Medicine, Mansoura University, with approval code R.24.08.2752 on 23/10/2024. A written informed consent was acquired from every participant. All methods were performed in accordance with the relevant guidelines and regulations.

Participants were enrolled from both in-and-outpatient clinics of the Internal Medicine Department, Rheumatology & Rehabilitation, and Physical Medicine Department, Mansoura University Hospitals. The study was conducted at Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Kasr Alainy Hostipals.

The Behcet’s Disease Current Activity Form (BDCAF) was used for assessment of behcet disease activity by examining the history and clinical symptoms caused by BD in the last four weeks using the following 12 components: headache, oral aphthous, genital ulcers, skin erythema, skin pustules, arthralgia, arthritis, nausea/vomiting/abdominal pain, diarrhea with altered/frank blood per rectum, eyes, central nervous system, and major blood vessels. Each component is given a score of absent (0) or present1. The BD activity index (BDCAF) is calculated by adding the scores of each component, with a maximum score of 12. A total score ≥ 2 is defined as active BD, whereas < 2 is defined as inactive BD15.

The inclusion criteria compromised female and male adult patients with a confirmed diagnoses of Behçet’s disease based on the international criteria for Behçet’s disease (ICBD), defined by the International Team for the Revision of ICBD16.

Patients with any other inflammatory disease or chronic infectious disorder were excluded in the study. Pregnant and lactating females, cognitively impaired, or mentally disabled subjects were also excluded from this study.

For the healthy control group, demographic data (age and sex) and selected clinical information, including family history and associated diseases/comorbidities, were recorded. Other clinical variables were not systematically collected, as the controls served as a reference group for biomarker comparison.

Molecular biology techniques for serum biomarkers

For each participants, a five mL blood sample was collected and centrifuged for serum separation to detect the expression and concentration levels of circRNA-FUNDC1 and TNF-α by quantitative PCR and ELISA (enzyme linked immunosorbent assay) respectively.

Expression level of circRNA-FUNDC1 by real-time qPCR (quantitative polymerase chain reaction)

By using Qiagen, total RNA was extracted from separated serum. Linear RNAs have been degraded using an exonuclease such as ribonuclease R (RNase R). RNase R from E. coli, is a 3′to 5’ exoribonuclease that can digest linear RNA. One µg of extracted RNA was incubated with 3 U of RNase R (LGC, Cat# RNR07250) (Sigma Aldrich, Germany**)** in 1X RNase R reaction buffer at 37 °C for 20 min, then heat inactivation at 70 °C for 10 min. Samples underwent RNA quantitation and purity evaluation using the ND-1000 NanoDrop (spectrophotometer (NanoDrop Technologies, Inc., Wilmington, USA). Afterward, a reverse transcription (RT) on total RNA was done in a final volume of 20uL using the miScript II RT kit (Qiagen, Valencia, CA, USA). For the detection of the expression level of circRNA-FUNDC1, quantitative real-time PCR (qRT-PCR) took place. Then, SYBER Green PCR kit was ued to detect the relative quantitative by using Qiagen, catalog 218073).

The sequence of the primers for circRNA-FUNDC1 (human circRNA-FUNDC1 forward primer: 5′-CCAGAGTCTCTAGGGCAAGG-3′, reverse primer: 5′ - TGCTTTGTTCGCTCGTTTCT-3′) were used for quantitative real time PCR. GAPDH (GAPDH forward, 5′-TGCACC ACC AAC TGC TTA GC-3′, reverse: 5′-GGC ATG GAC TGT GGT CAT GAG-3′ (Ref Seq: NM_001256799.3) were used as reference gene. Ct of GAPDH was subtracted from the values of Ct of circRNA-FUNDC1 for all participants. Then Ct values was performed by subtracting the control Ct values from the patients’ values. The relative quantitation or fold changes (FC) were calculated for the circular RNA using the method of 2−ΔΔCt. After the completion of qRT-PCR cycles, melting curves were analyzed to confirm the targeted circRNA-FUNDC1 expression.

Detection of the concentration level of TNF-α using ELISA

For measuring the concentration level of TNF-α in serum, enzyme-linked immunosorbent assay (ELISA) was used (BT LAB ELISA, Catalog no. E0082Hu).

Statistical analysis of data

On windows 8.1 and by using Statistical Package of Social Science (SPSS) software version 22.0 data was analyzed. Kruskal-Wallis test for comparing groups having quantitative variables was used. Mean average, standard deviation (SD), and range were used for quantitative data. Frequency distribution and percentages were used for qualitative data. Independent Student t-test was used for the analysis of parametric data, while for nonparametric data Chi-square test (χ2) was used. Multiple pairwise comparisons were performed where the Benjamini-Hochberg false discovery rate (FDR) procedure was applied to control for Type I error. Pearson correlation test was used for correlating quantitative parameters at a significance of ≤0.05. To further evaluate potential treatment-related confounding while minimizing multicollinearity, additional multivariate models included only medications that demonstrated significant associations in the univariate analyses by which regression coefficients (β), 95% confidence intervals (CI) and coefficients of determination (R²) were reported.