The influence of genetic polymorphisms in TLR4 and TIRAP, and their expression levels in peripheral blood, on susceptibility to sepsis

Abstract

The present study aimed to investigate whether genetic polymorphisms in the Toll-like receptor (TLR)-4 and Toll/interleukin-1 receptor (TIR)-associated protein (TIRAP) genes, and/or their expression levels, influence the susceptibility of a patient to sepsis. A total of 106 patients with sepsis were divided into two groups on the basis of their acute physiology and chronic health evaluation (APACHE) II scores: i) Sepsis group A (APACHE II <20) and ii) Sepsis group B (APACHE II >20). In addition, 100 healthy volunteers were enrolled into the control group. Polymerase chain reaction-restriction fragment length polymorphism assay was used to detect the following genetic polymorphisms: The Ser180Leu allele of the TIRAP gene and the Asp299Gly and Thr399I1e alleles of the TLR4 gene. Furthermore, the protein expression levels of TLR4 and TIRAP were analyzed using an enzyme-linked immunosorbent assay. Genetic polymorphisms were not detected for the TLR4 and TIRAP genes; however, the protein expression levels of TLR4 and TIRAP differed significantly between the control, sepsis A and sepsis B groups (P<0.01). An APACHE II score of 20 was used as a baseline in order to differentiate sepsis severity. Pearson analysis demonstrated that TLR4 and TIRAP protein expression levels were positively correlated with sepsis severity (r=0.931 and 0.972; P<0.05), and TLR4 protein expression levels were positively correlated with those of TIRAP (r=0.936; P<0.05). The results of the present study suggested that the protein expression levels of, but not genetic polymorphisms in, TLR4 and TIRAP were associated with the severity of sepsis.

Keywords: Toll-like receptor 4; Toll/interleukin-1 receptor-associated protein; genetic polymorphisms; sepsis.

Figure 1.

(A) Electrophoresis of polymerase chain reaction (PCR) products. Lane 1, DNA Marker I (100, 200, 300, 400, 500 and 600 bp, from top-to-bottom); lane 2, positive control; lanes 3, 6, 9 and 12, Asp299Gly (TLR4) gene (218 bp); lanes 4, 7, 10 and 13, Thr399Ile (TLR4) gene (405 bp); lanes 5, 8, 11 and 14, Ser180Leu (TIRAP/Mal) gene (161 bp). (B) Electrophoresis of purified PCR products. Lane M, DNA Marker 20 (200, 300, 400 and 500 bp, from top-to-bottom); lanes 1, 4 and 7, Asp299Gly (TLR4) gene (218 bp); lanes 2, 5 and 8 Thr399Ile (TLR4) gene (405 bp); lanes 3, 6 and 9 Ser180Leu (TIRAP/Mal) gene (161 bp).

Figure 2.

Electrophoresis of polymerase chain reaction products following digestion with restriction enzymes. (A) Toll-like receptor (TLR)4 Asp299Gly allele digested with the NcoI restriction enzyme; (B) TLR4 Thr399Ile allele digested with the HindI restriction enzyme; and (C) Toll/interleukin-1 receptor-associated protein Ser180Leu allele digested with the Eam1105I restriction enzyme. Lane M, DNA Marker I (20, 80, 100, 200, 400 and 600 bp, from top-to-bottom). Lanes 1–8 correspond to the numbers of the samples.

Figure 3.

Representative sequence diagram of the Toll-like receptor 4 (A) Asp299Gly and (B) Thr399Ile alleles, and the (C) Toll/interleukin-1 receptor-associated protein Ser180Leu gene.

Figure 4.

Sequencing results of the Toll-like receptor (TLR)4 (Asp299Gly) gene using Chromas 2.31 software. TLR4-04 and −05 correspond to sequences from the control group, whereas all others correspond to sequences from the sepsis groups.

Figure 5.

Sequencing results of the Toll-like receptor (TLR)4 (Thr399I1e) gene using Chromas 2.31 software. TLR4-04 and −05 correspond to sequences from the control group, whereas all others correspond to sequences from the sepsis groups.

Figure 6.

Sequencing results of the Toll/interleukin-1 receptor-associated protein (TIRAP) Ser180Leu allele using Chromas 2.31 software. TIRAP-04 and −05 correspond to the control group, whereas all others correspond to sequences from the sepsis groups.