Evaluation of tumor necrosis factor (TNF)-α mRNA expression level and the rs1799964 polymorphism of the TNF-α gene in peripheral mononuclear cells of patients with inflammatory bowel diseases

Abstract

Crohn's disease (CD) and ulcerative colitis (UC) are types of chronic inflammatory bowel disease (IBD) of which the actual causes remain unknown. Emerging data indicate that alterations in cytokine synthesis may be involved in IBD pathogenesis. The aim of the present study was to determine whether the tumor necrosis factor (TNF)-α mRNA expression level and rs1799964 polymorphism are the genetic susceptibility component of IBD development. The TNF-α mRNA expression level of peripheral blood mononuclear cells (PBMCs) was measured using comparative reverse-transcription quantitative polymerase chain reaction (PCR). Genomic DNA from 201 individuals (CD: n=15; UC: n=86; control subjects: n=100) was analyzed for the presence of the TNF-α-1031 polymorphism by PCR-restriction fragment length polymorphism. An increased TNF-α mRNA expression level was additionally observed in the CC genotype of the -1031 TNF-α gene polymorphism compared with the TC and TT genotypes (P<0.05). Furthermore, the present results revealed that there was no significant difference in the genotype/allele frequencies of the -1031 TNF-α gene polymorphism in Iranian IBD patients. By comparison, the TNF-α mRNA expression level was evaluated in patients with a history of taking medications and demonstrated a significant association in the group that received the 5-ASA + Pred + AZA,5. 5-ASA + Pred + AZA + IFX when compared with the other groups (P<0.05). Thus, these results support the hypothesis that overexpression of the TNF-α gene, which correlated with the CC genotype, may represent a genetic risk factor for Iranian IBD.

Keywords: Crohn's disease; TNFα-1031 T>C polymorphism; inflammatory bowel disease; quantitative polymerase chain reaction; ulcerative colitis.

Figure 1.

Polymorphism fragments on agarose gel following digestion with BbsI restriction enzyme. Ladder 16–21, 24, 25, 30: TT; ladder 22, 23, 29: TC; ladder 27: CC; L50, ladder 50 bp. RFLP, restriction fragment length polymorphism.

Figure 2.

TNF-α mRNA expression level (A) at different IBD pathological phases, (B) according to the type of IBD (UC or CD) and (C) according to drug history. (D) Interaction between the −1031 genotype and TNF-α expression level. TNF-α, tumor necrosis factor-α; IBD, inflammatory bowel disease; UC, ulcerative colitis; CD, Crohn's disease; RQ, relative quantification; 5-ASA, 5-aminosalicylic acid; Pred, prednisone; AZA, azathioprine; IFX, infliximab.