The study group consisted of 146 IBD Zhuang patients without genetic kinship enrolled in the Gastroenterology Department, First Affiliated Hospital of Guangxi Medical University, from February 2007 to October 2011. The control group included 164 healthy Zhuang subjects without genetic kinship who were healthy individuals or patients with functional dyspepsia, and did not have liver or gastrointestinal diseases. All patients had a well-established diagnosis of UC or CD, according to standard clinical criteria based on endoscopic and histopathological examinations. There were no significant differences in age and sex between the study group and the control group. All patients and healthy controls gave informed consent and the study was approved by the ethical committee of the institute.

Genomic DNA was extracted according to the modified protocol of Taggart. The primers used to amplify the TLR4 gene (Asp299Gly, Thr399 Ile) were designed according to the National Center for Biotechnology Information gene database, NM_138554, and the TLR2 gene (Arg677Trp, Arg753Gln) with NM _003264 shown in Table 1 (primers were synthesized in SHENGGONG Biological Technology Co., Ltd., Shanghai, China).

Amplification was performed using H₂O₂ 10.5 μL, 2 × Taq polymerase chain reaction (PCR) Master-mix 11.5 μL (TIANGEN), DNA 1 μL, and 1 μL in addition to 10 μmol/L of each primer in a total volume of 25 μL. For Asp299Gly and Arg753Gln, cycle conditions were an initial denaturation for 5 min at 95 °C, followed by 28 cycles of denaturing at 95 °C for 40 s, annealing at 58 °C for 30 s, primer extension at 72 °C for 50 s, followed by a final extension at 72 °C for 10 min. For Thr399Ile and Arg677Trp, cycle conditions were an initial denaturation for 5 min at 95 °C, followed by 28 cycles of denaturing at 95 °C for 40 s, annealing at 62 °C for 40 s, primer extension at 72 °C for 50 s, followed by a final extension at 72 °C for 10 min (Thermo Electron Corporation, Waltham, MA, United States). All the PCR products were electrophoresed on a 1.5% agarose gel, with 1 × tris-borate-EDTA buffer, V = 100V for 30 min, and visualized under ultraviolet illumination (Bio-Rad Gel Doc-2000, United States).

Five μL PCR products of TLR4 gene (Asp299Gly, Thr399Ile) and TLR2 gene (Arg677Trp，Arg753Gln) were digested at 37 °C overnight with 0.5 μL (10 U) Nco I, Hinf I, Aci I, and Pst I restriction enzymes (Fermentas), respectively. After enzymatic digestion, the fragments were separated and visualized by gel electrophoresis (2% Yito Bio-Instrument Company Ltd., Shanghai, China).

PCR products of each SNP were purified with a PCR purification kit (QIAGEN, Hilden, Germany) and were sequenced using an ABI Prism 377 DNA sequencer (LIU HE HUA DA GENE Technology Co, Ltd., Beijing, China).

The genetic equilibrium was tested using Hardy-Weinberg. Allele and genotype frequencies in patients and in controls were compared using the χ² test with SPSS 13.0, and P values were considered significant at a level of < 0.05.

Nco I restriction enzyme identified the sequence of CCATGG. For the heterozygous type (AG), this could be generated when digested with Asp299Gly and three bands of 249 bp, 223 bp and 26 bp, where one chain was cut, and the others were not. For the homozygous type (GG), a transite to G, Nco I identified the mutated site, and the mutated DNA was visible as a double band of 223 bp and 26 bp, whereas a single band of 249 bp was observed in the wild type (AA), as the site was not cut. In this study, only the wild type (AA) was detected, and no other types (Figure 1A).

Hinf I restriction enzyme identified the sequence of GANTC. For the heterozygous type (CT), this could be generated when digested with Thr399Ile and three bands of 406 bp, 377 bp and 29 bp, where only one chain was cut, and the others were not. For the homozygous type (TT), C transite to T, Hinf I identified the mutated site, and the mutated DNA was visible as a double band of 377 bp and 29 bp, whereas a single band of 406 bp was observed in the wild type (CC), as the site was not cut. In this study, only the wild type (CC) was detected, and no other types (Figure 1B).

Aci I restriction enzyme identified the sequence of CCGC. For the heterozygous type (CT), this could be generated when digested with Arg677Trp and three bands of 199 bp, 124 bp and 75 bp, where one chain was cut, and the others were not. For the homozygous type (TT), C transite to T, the site was not cut by Aci I restriction enzyme, and a single band of 199 bp was observed, for the wild type (CC), it identified the site, and the DNA was visible as a double band of 124 bp and 75 bp. In this study, only the wild type (CC) was detected, and no other types (Figure 1C).

Pst I restriction enzyme identified the sequence of CTGCAG. For the heterozygous type (GA), this could be generated when digested with Arg753Gln and three bands of 254 bp, 214 bp and 40 bp, where one chain was cut, and the others were not. For the homozygous type (AA), G transite to A, Pst I identified the mutated site, and the mutated DNA was visible as a double band of 214 bp and 40 bp, whereas a single band of 254 bp was observed in the wild type (GG), as the site was not cut. In this study, only the wild type (GG) was detected, and no other types (Figure 1D).

The pathogenesis of inflammatory bowel disease (IBD) is not completely clear, however, contributing factors may include immunology, genetics, environment and infection. It has been reported that the NOD2/CARD15 polymorphisms (single nucleotide polymorphisms, SNPs) were found to be associated with Crohn’s disease (CD) in Caucasian populations. However, the SNPs were not found to be associated with CD in Japan and China. In addition, the TLR2 and TLR4 were reported to provide evidence for several determinants. The study assessed whether the known gene polymorphisms in the toll-like receptor 2 (TLR2) and TLR4 genes determined susceptibility for IBD in the Guangxi Zhuang population. Since Guangxi has a large Zhuang population, genetic diseases and gene polymorphisms are unique.

TLR4 (Asp299Gly, Thr399Ile) and TLR2 (Arg677Trp, Arg753Gln) were found to be associated with IBD in Tunisia, Germany and Spain, but not in Hong Kong, Hubei, Zhengjiang, Shanghai and Japan. This is possibly due to the existence of racial and geographic differences. This study determined whether the TLR4 (Asp299Gly, Thr399Ile) and TLR2 (Arg677Trp, Arg753Gln) were associated with IBD in the Guangxi Zhuang population from the Guangxi Zhuang Autonomous Region of China.

Only a few studies have investigated the TLR2 and TLR4 gene polymorphisms in China. Moreover, all the subjects in these studies were from the Han population. This is the first study to report that TLR4 gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg753Gln polymorphisms may not be associated with IBD in the Zhuang population from the Guangxi Zhuang Autonomous Region of China.

TLR2 and TLR4 variants may be rare or non-existent in the Zhuang population from the Guangxi Zhuang Autonomous Region of China.

TLRs play a key role in microbial recognition in innate immunity and control the adaptive immune responses. Among the TLRs, TLR2 recognizes bacterial components such as lipoprotein, lipoteichoic acids, peptido-glycan and zymozan. TLR4 requires CD14 to form the lipopolysaccharide (LPS) receptor. LPS, found in the outer membrane of Gram-negative bacteria, is opsonized by LPS-binding protein, and recognized by CD14. The LPS-LPS binding protein-CD14 complex activates TLR4, which results in the activation of NF-κB. TLR2 and TLR4 gene mutations or deletions can induce abnormal immune responses.

This study demonstrates no association of TLR2 and/or TLR4 polymorphism with IBD in a patient cohort within a restricted chinese population. Although this is a “negative” result, it adds to the impact of genetic and/or ethnical predisposition to IBD.