Background and Aim: Toll-like receptor (TLR) 2 and TLR4 are cell surface signaling receptors that are involved in the recognition of and host response to <i>Helicobacter pylori</i>. Our aim was to investigate the association between TLR gene polymorphisms and susceptibility of Japanese subjects to 4 H. pylori-related gastrointestinal diseases. Methodology: A total of 100 patients with histologically diagnosed gastric cancer, 105 patients with gastric ulcer, 102 with atrophic gastritis, 72 with duodenal ulcer and 428 healthy controls were recruited. A TaqMan assay was used to genotype 7 single nucleotide polymorphisms (SNPs) in TLR2 and 6 SNPs in TLR4. Results: There was a tendency for TLR4 rs10759932 TC/CC genotypes to be associated with a decreased risk of gastric cancer (p = 0.059); however, this did not reach statistical significance. No significant associations were found between polymorphisms in TLR2 or TLR4 and the risks for gastric cancer, gastric ulcer, duodenal ulcer, or atrophic gastritis. Conclusion: The 13 SNPs inTLR2 and TLR4 examined in this study may not be linked with the development of H. pylori-related gastrointestinal diseases. Further studies with larger numbers of subjects are necessary to verify the present findings.
Helicobacter pylori is a spiral-shaped, microaerophilic, gram-negative bacterium that colonizes the human stomach and establishes persistent infection in the gastric mucosa. Numerous studies have also shown that H. pylori is a risk factor for development of upper gastrointestinal diseases, such as peptic ulcer disease, atrophic gastritis, gastric mucosa-associated lymphatic tissue lymphoma and gastric cancer [
Toll-like receptors (TLRs) are proteins that play important roles in pattern recognition of various substances that are produced by microorganisms. To date, 10 different TLRs have been identified in humans [
TLR2 and TLR4 are expressed on several types of cells in many human tissues, and they act as important mediators of the inflammatory response in the first line of host defense against microbial infections. Amongst other cell types, these receptors are expressed on gastric epithelial cells and are involved in the recognition of H. pylori and in the immunological response of the host to these bacteria [
Recent evidence has suggested that polymorphisms in genes encoding TLR are associated with several cancers, such as colorectal cancer, cervical cancer and gallbladder cancer [
This study protocol was approved by the Ethics Committee of Tokai University Hospital, Kanagawa, Japan. Written informed consent was obtained from all individuals prior to enrolling them in the study, according to the ethical guidelines of the Declaration of Helsinki. A total of 100 patients with histologically diagnosed gastric cancer (male: 79 cases, age: 72.3 ± 8.2 y [mean ± SD]), 105 patients with gastric ulcer (male: 71 cases, age: 66.3 ± 13.6 y), 102 patients with atrophic gastritis (male: 41 cases, age: 69.6 ± 9.4 y), and 72 patients with duodenal ulcer (male: 51 cases, age: 59.0 ± 14.7 y) were recruited at Tokai University Hospital. The cancer-free control group comprising individuals with no previous history of gastric disease, was composed of 428 healthy individuals (male: 255 cases, age: 48.4 ± 13.12 y), recruited at Tokai University School of Medicine.
Peripheral blood samples were obtained from all subjects and genomic DNA was subsequently extracted from the samples using a DNA extraction kit (Takara, Otsu, Japan).We selected tagging SNPs covering theTLR2 and TLR4 regions, using the HapMap Japanese data set (MAF ≥ 5%, pairwiser2 ≥ 0.8, PHWE ≥ 0.05). In addition, we selected non-synonymous SNPs that had previously been reported to be associated with other diseases. A TaqMan 5’ exonuclease assay was used to genotype 7 SNPs in TLR2 and 6 SNPs in TLR4 with primers supplied by Applied Biosystems (Foster City, CA, USA), following the manufacturer’s instructions. The fluorescence signal of the probes was detected using a 755 Real-Time PCR System (Applied Biosystems). 7 SNPs in TLR2 and 6 SNPs in TLR4 were genotyped in a total of 379 patients and controls.
Differences in allele and genotype frequencies among the patients with either of 4 H. pylori-related gastroduodenal diseases and the controls were assessed by chi-square tests. The chi-square test was also used for determining Hardy-Weinberg equilibrium. Continuous variables were calculated as the mean ± standard deviation (SD). Genotype and allele frequencies were analyzed using logistic regression models adjusted for age and gender. Odds ratios (ORs) were calculated using a dominant model (i.e., combining heterozygous and homozygous for the minor allele) for all polymorphisms. Differences in continuous variables were analyzed using the Mann-Whitney U-test. Statistical analyses were performed using the Stat Flex (version 6.0) computer program. The criterion for statistical significance was a p-value of < 0.05.
The genotype and allele frequency distributions of 9 polymorphisms were found to be in Hardy-Weinberg equilibrium, in both control and gastroduodenal disease groups (data not shown). TLR2 rs5743704, TLR2 rs5743708, TLR4 rs4986790, and TLR4 rs4986791 polymorphisms were not informative (heterozygous) in either control or patient groups.
The genotype and allele frequencies of the 9 informative polymorphisms and OR values of gastroduodenal disease patients and the control groups, adjusted for age and gender, are shown in
Responses to TLR ligands may be altered by SNPs in TLR encoding genes, resulting invariable susceptibility to H. pylori-related gastrointestinal diseases after long-term infection. Therefore, an investigation of the association between polymorphisms in TLR2 and TLR4 and the risks of H. pylori-related gastrointestinal diseases is of value. This study involved an exhaustive genetic analysis, exploring the association between SNPs in TLR2 and TLR4 and susceptibility to H. pylori-related gastroduodenal diseases, including gastric cancer.
Previous studies focused only on 1 or 2 SNPs in these genes, which may not have been adequate for determining their potential effects on susceptibility to gastroduodenal diseases. The SNPs most intensively investigated in TLR4 are rs4986790 and rs4986791, which had been shown to be associated with susceptibility to gastric cancer in Caucasian, Brazilian, and Indian populations [
We found weak association between a polymorphism in the 5’-flanking region of TLR4 (rs10759932) and the risk of gastric cancer; however, this association failed to reach statistical significance. There have been only few studies that evaluated the association between this TLR4 polymorphism and the risks of gastrointestinal diseases. In a recent Chinese study, Fan et al. reported that TLR4 rs10759932 TC and CC genotypes were associated with a lower risk of developing precancerous lesions of the stomach [
Polymorphisms Genotypes/Alleles | Control N = 428 n (%) | Patients | |||||||
---|---|---|---|---|---|---|---|---|---|
Gastric Cancer | Gastric Ulcer | Atrophic Gastritis | Duodenal Ulcer | ||||||
N = 100 n (%) | OR (95% CI), p | N = 105 n (%) | OR (95% CI), p | N = 102 n (%) | OR (95% CI), p | N = 72 n (%) | OR (95% CI), p | ||
TLR2 rs7696323 C/C C/T + T/T C T | 251 (59.3) | 51 (51.0) | 1.40 (0.75 - 2.62), 0.295 | 68 (64.8) | 0.78 (0.47 - 1.32), 0.360 | 64 (62.7) | 0.68 (0.36 - 1.31), 0.260 | 38 (53.8) | 1.15 (0.68 - 1.94), 0.600 |
172 (40.7) | 49 (49.0) | 37 (35.2) | 38 (37.3) | 34 (46.2) | |||||
0.76 | 0.72 | 1.29 (0.79 - 2.12), 0.311 | 0.71 | 0.74 (0.48 - 1.15), 0.177 | 0.78 | 0.72 (0.42 - 1.24), 0.240 | 0.73 | 1.06 (0.70 - 1.62), 0.770 | |
0.24 | 0.28 | 0.29 | 0.22 | 0.27 | |||||
TLR2 rs1898830 A/A A/G + G/G A G | 123 (29.0) | 29 (29.0) | 1.07 (0.53 - 2.13), 0.859 | 35 (33.3) | 0.96 (0.56 - 1.65), 0.887 | 32 (31.4) | 1.31 (0.64 - 2.67), 0.460 | 26 (36.1) | 0.88 (0.51 - 1.54), 0.660 |
301 (71.0) | 71 (71.0) | 70 (66.7) | 70 (68.6) | 46 (63.9) | |||||
0.54 | 0.56 | 1.02 (0.65 - 1.60), 0.931 | 0.49 | 1.18 (0.83 - 1.69), 0.354 | 0.53 | 1.29 (0.82 - 2.04), 0.276 | 0.61 | 0.89 (0.61 - 1.30), 0.557 | |
0.46 | 0.44 | 0.51 | 0.47 | 0.39 | |||||
TLR2 rs3804099 T/T T/C + C/C T C | 213 (50%) | 55 (55.0) | 0.73 (0.39 - 1.37), 0.325 | 51 (48.6) | 1.00 (0.61 - 1.66), 0.990 | 54 (52.9) | 0.87 (0.46 - 1.65), 0.680 | 30 (41.6) | 1.27 (0.75 - 2.15), 0.380 |
213 (50%) | 45 (45.0) | 54 (51.4) | 48 (47.1) | 42 (58.4) | |||||
0.69 | 0.75 | 0.67 (0.41 - 1.10), 0.111 | 0.68 | 0.91 (0.62 - 1.34), 0.640 | 0.7 | 0.87 (0.54 - 1.41), 0.569 | 0.65 | 1.09 (0.73 - 1.60), 0.682 | |
0.31 | 0.25 | 0.32 | 0.3 | 0.35 | |||||
TLR2 rs3804100 T/T T/C + C/C T C | 229 (53.8) | 60 (60.0) | 0.73 (0.39 - 1.39), 0.340 | 54 (51.4) | 1.10 (0.66 - 1.82), 0.713 | 56 (54.9) | 0.92 (0.49 - 1.75), 0.800 | 33 (45.8) | 1.30 (0.78 - 2.19), 0.340 |
197 (46.2) | 40 (40.0) | 51 (48.6) | 35 (45.1) | 39 (54.2) | |||||
0.72 | 0.78 | 0.64 (0.38 - 1.07), 0.088 | 0.71 | 0.96 (0.65 - 1.42), 0.827 | 0.72 | 0.90 (0.55 - 1.48), 0.686 | 0.68 | 1.10 (0.72 - 1.59), 0.740 | |
0.28 | 0.25 | 0.29 | 0.28 | 0.32 | |||||
TLR2 rs7656411 G/G G/T + T/T G T | 141 (33.1) | 26 (26.0) | 1.48 (0.75 - 2.93), 0.258 | 34 (32.4) | 1.20 (0.70 - 2.05), 0.506 | 33 (32.4) | 1.79 (0.89 - 3.59), 0.100 | 29 (40.2) | 0.86 (0.50 - 1.48), 0.590 |
285 (66.9) | 74 (74.0) | 71 (67.6) | 69 (67.6) | 43 (59.8) | |||||
0.57 | 0.53 | 1.08 (0.69 - 1.70), 0.722 | 0.59 | 0.93 (0.65 - 1.33), 0.674 | 0.57 | 1.12 (0.71 - 1.77), 0.614 | 0.61 | 0.86 (0.59 - 1.26), 0.441 | |
0.43 | 0.47 | 0.41 | 0.43 | 0.39 | |||||
TLR4 rs10759932 T/T T/C + C/C T C | 236 (56.1) | 68 (68.0) | 0.52 (0.26 - 1.02), 0.059 | 59 (56.2) | 0.97 (0.58 - 1.61), 0.905 | 61 (59.8) | 1.03 (0.54 - 1.97), 0.930 | 41 (56.9) | 0.97 (0.57 - 1.64), 0.900 |
185 (44.9) | 32 (32.0) | 46 (43.8) | 41 (40.2) | 31 (43.1) | |||||
0.75 | 0.81 | 0.76 (0.43 - 1.33), 0.333 | 0.74 | 1.09 (0.72 - 1.64), 0.691 | 0.77 | 1.17 (0.69 - 1.99), 0.554 | 0.76 | 1.03 (0.67 - 1.58), 0.906 | |
0.25 | 0.19 | 0.26 | 0.23 | 0.24 |
TLR4 rs1927911 G/G G/A + A/A G A | 174 (40.9) | 50 (50.0) | 0.91 (0.48 - 1.70), 0.758 | 44 (41.9) | 1.26 (0.75 - 2.12), 0.373 | 48 (47.1) | 1.43 (0.75 - 2.75), 0.270 | 27 (37.5) | 1.42 (0.82 - 2.44), 0.210 |
---|---|---|---|---|---|---|---|---|---|
251 (59.1) | 50 (50.0) | 61 (58.1) | 54 (52.9) | 45 (62.5) | |||||
0.64 | 0.7 | 0.95 (0.59 - 1.52), 0.820 | 0.66 | 1.08 (0.75 - 1.57), 0.678 | 0.69 | 1.16 (0.72 - 1.88), 0.537 | 0.6 | 1.28 (0.88 - 1.88), 0.197 | |
0.36 | 0.3 | 0.34 | 0.31 | 0.4 | |||||
TLR4 rs11536889 G/G G/C + C/C G C | 243 (57.2) | 53 (53.0) | 1.02 (0.54 - 1.92), 0.952 | 64 (61.0) | 0.77 (0.46 - 1.30), 0.329 | 55 (53.9) | 0.75 (0.39 - 1.43), 0.390 | 47 (65.2) | 0.66 (0.38 - 1.14), 0.130 |
182 (42.8) | 47 (47.0) | 41 (39.0) | 47 (46.1) | 25 (34.8) | |||||
0.75 | 0.74 | 0.97 (0.58 - 1.61), 0.905 | 0.79 | 0.80 (0.52 - 1.23), 0.309 | 0.75 | 0.82 (0.48 - 1.38), 0.447 | 0.81 | 0.67 (0.42 - 1.07), 0.095 | |
0.25 | 0.26 | 0.21 | 0.25 | 0.19 | |||||
TLR4 rs7037117 A/A A/G + G/G A G | 269 (63.3) | 63 (63.0) | 1.33 (0.69 - 2.56), 0.390 | 66 (62.9) | 1.25 (0.74 - 2.09), 0.407 | 68 (66.7) | 1.22 (0.62 - 2.38), 0.570 | 41 (56.9) | 1.49 (0.87 - 2.54), 0.150 |
156 (36.7) | 37 (37.0) | 39 (37.1) | 34 (33.3) | 31 (43.1) | |||||
0.8 | 0.8 | 1.29 (0.74 - 2.23), 0.371 | 0.8 | 1.18 (0.76 - 1.83), 0.456 | 0.83 | 1.12 (0.63 - 1.99), 0.710 | 0.76 | 1.38 (0.89 - 2.14), 0.152 | |
0.2 | 0.2 | 0.2 | 0.17 | 0.24 |
stomach. The increased TLR activity may then modulate the development of gastric cancer. Since the possibility that TLR4 may be involved in the development of gastric cancer cannot be ruled out in the Japanese population, further studies employing a larger number of subjects are necessary.
Achyut et al. [
A 22-bp insertion/deletion polymorphism, known as TLR2 -196 to -174 deletion, has been reported to influence the transcription activity of TLR2. Recent evidence has suggested that this polymorphism is associated with a variety of cancers, such as gastric cancer, prostate cancer, cervical cancer, and so on [
The present study had several limitations. The H. pylori status of participants was not examined and we could not investigate the association between polymorphisms in TLR2 or TLR4 and gastric atrophy in more detail, because of a lack of serum samples.
In conclusion, this study revealed that SNPs in TLR2 and TLR4 may not be associated with the development of H. pylori-related gastroduodenal diseases, and did not reproduce the previous noted association between gastric cancer and TLR4 polymorphisms in the Japanese population. However, weak association was found between a polymorphism in the 5’-flanking region of TLR4 (rs10759932) and the risk of gastric cancer. Since it remains possible that TLR4 signaling pathways can play a role in the development of gastric cancer, further well-designed, comprehensive studies, employing a larger number of subjects, are necessary to verify our findings.
The authors declare no conflicts of interest.