TNF-alpha gene 1031 T/C polymorphism in Turkish patients with Behçet's disease.

BACKGROUND: Genetic factors that predispose individuals to Behçet's disease (BD) are considered to play an important role in development of the disease. The tumour necrosis factor (TNF)-alpha gene, which is closely linked to the HLA-B51 gene, is involved in susceptibility for BD. Recently, a polymorphism at position -1031 within the TNF-alpha promoter region was demonstrated to be responsible for susceptibility to BD in a British population. However, the functional effects of this polymorphism have not yet been determined.
OBJECTIVES: To investigate the possible relation of the TNF-alpha-1031 T/C polymorphism with susceptibility to BD in a Turkish population and to determine the functional importance of this polymorphism.
METHODS: Ninety-nine unrelated patients (47 women, 52 men; mean +/- SD age, 34.10 +/- 10.53 years) with BD and 103 ethnically matched healthy controls (52 males, 51 females; mean +/- SD age, 40.25 +/- 14.15) were enrolled in the study. For genotyping, polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis was employed. The functional importance of TNF-alpha-1031 T/C polymorphism was determined with an enzyme-linked immunospot (ELISPOT) assay. For this purpose, mononuclear cells obtained from BD patients and controls were analysed for TNF-alpha and interferon (IFN)-gamma production.
RESULTS: A significant difference was observed between BD patients and controls with respect to the allele frequency of TNF-alpha-1031C [P = 0.018, OR = 1.83, 95% confidence interval (CI) = 1.07-3.13]. When the allele frequencies were analysed according to the clinical features, the T allele in patients with positive skin pathergy test (SPT) was significantly increased when compared with those of patients without these findings (P = 0.004, OR = 2.75, 95% CI = 1.3-5.86). To demonstrate the frequency of TNF-alpha and IFN-gamma producing cells, mononuclear cells from four representative individuals of each genotype were used and the spontaneous and stimulated TNF-alpha and IFN-gamma values (spot numbers) were analysed. Compared with the control groups, a significant increase was observed in the number of cells producing TNF-alpha obtained from BD patients (P < 0.001). Moreover, the stimulation index for TNF-alpha [bacterial lipopolysaccharide (LPS) stimulated/unstimulated] was higher for the CC genotype (9 +/- 9.5) with respect to the other genotypes (TT; 1.3 +/- 0.3 and TC; 1.2 +/- 0.2). While the difference in the spontaneous IFN-gamma values between groups were not statistically significant, the stimulated IFN-gamma values were found to be significantly increased in the BD group when compared with the healthy control group (P = 0.004).
CONCLUSIONS: Our results showed that, in the Turkish population the TNF-alpha-1031C allele is associated with susceptibility to BD. On the other hand, carrying the T allele may render patients more prone to developing a positive skin pathergy test. In addition, ELISPOT assays revealed that BD patients exhibited a significantly higher number of mononuclear cells producing TNF-alpha, and cells obtained from patients with a CC genotype had a stronger response to LPS stimulation. The strong IFN-gamma response upon LPS stimulation in BD patients supports the previous findings that BD is a Th1 driven disease. These findings suggest that the TNF-alpha-1031 polymorphism may have a functional effect and could explain the reason for high levels of TNF-alpha production observed in BD patients.