Abstract
Background/Aims
Genetic polymorphisms in Toll-like receptors (TLRs) are important influence on gastric lesion development and Helicobacter pylori susceptibility.
Materials and Methods
TLR2 rs3804099 and rs3804100 and TLR4 rs10759932 were determined in a total of 400 patients. The association among genotypes and the risk of gastric lesion development and H. pylori susceptibility were evaluated by the odds ratios (ORs) and 95% confidence intervals (95% CIs) from logistic regression analyses.
Results
TLR4 rs10759932, C/C homozygous genotype was associated with an increased risk of premalignant/malignant (OR=2.48, 95% CI=1.96–4.62, p=0.015). The recessive model of TLR4 rs10759932 showed a decreased risk of H. pylori susceptibility (adjusted OR=0.52, 95% CI=0.38–0.82, p=0.046). Meanwhile, the recessive model was associated with an increased risk of non-malignant (OR=3.46, 95% CI=2.25–5.67, p=0.001). In subjects with H. pylori infection, the recessive model was associated with an increased risk of non-malignant (OR=2.28, 95% CI=1.24–3.57, p=0.001) and premalignant/malignant (OR=1.83, 95% CI=1.16–2.84, p=0.027).
Conclusion
TLR4 rs10759932, but not TLR2 rs3804099 and rs3804100, was associated with risk of premalignant and/or malignant and H. pylori susceptibility. H. pylori infection seems to contribute to chronic gastritis, and premalignant/malignant supported the development of the premalignant/malignant lesions involved in H. pylori infection that is critical to gastric cancer in Thai patients.
Keywords: Gastric cancer, Toll-like receptor 2, Toll-like receptor 4, Genetic polymorphisms, Helicobacter pylori
INTRODUCTION
Colonization of Helicobacter pylori in a human stomach causes chronic infection. Inflammation in the gastric mucosa induces the development of peptic ulcer. Patients with chronic active gastritis progress to atrophy gastritis and intestinal metaplasia due to inflammation in the gastric mucosa that can trigger gastric cancer (GC) (1,2). However, 15%–30% of patients with H. pylori-infected chronic gastritis develop peptic ulcerations, distal gastric adenocarcinoma, or gastric lymphoma (3,4).
With genetic heterogeneity, host factors are important in gastric carcinogenesis due to H. pylori infection, Toll-like receptors (TLRs), or host molecule response to pathogen-associated molecular patterns that bind to the spectrum of ligands (5,6). TLR recognition plays a crucial role in the defense against infection and immune system regulation. Thus, polymorphisms in the TLR genes affect host susceptibility to infection (7). TLR polymorphism might cause an imbalance of pro- and anti-inflammatory cytokine responses and modulate immune pathogenesis and cancer. TLRs detect endogenous ligands released of damaged tissues, necrotic cells, or cancer cells (6). Today, genetic polymorphisms in TLRs are associated with H. pylori-related diseases, H. pylori susceptibility, and inflammation (8).
TLR2 and TLR4 are implicated in the recognition of various bacterial cell wall components, such as lipopolysaccharide, peptidoglycans, and lipoproteins. Numerous studies of TLR2 and TLR4 polymorphisms are associated with H. pylori infection and GC. They report that impaired TLR function and TLR signaling pathways may result in an elevated risk of infection including occurrence of various pathologies and cancer (9–12). Additionally, TLR4 polymorphisms are associated with modified immune responses of the gastric mucosa (13–15) and substantially contribute to GC (16,17). However, in Thailand, no study investigating the role of the TLR2 and TLR4 polymorphisms on the H. pylori-related pathological process and the impact of TLR polymorphisms on the H. pylori-related gastric lesions including non-malignant, premalignant, and malignant has yet been established in Thai patients. The investigation of polymorphisms in the Thai population could provide novel insights into targeted treatment in genetically susceptible individuals and improve the prevention of H. pylori-related GC. Therefore, the study was aimed to evaluate the association of TLR2 rs3804099 and rs3804100 and TLR4 rs10759932 polymorphisms and the risk of H. pylori susceptibility as well as gastric mucosal pathology by affecting the variant genotype. The results could provide details of the association of genetic polymorphisms and H. pylori infection and gastric mucosal pathology in Thai patients.
MATERIALS AND METHODS
Patients
A total of 400 patients who received esophagogastroduodenoscopy (EGD) to investigate chronic abdominal pain participated in the study conducted from December 2014 to March 2016. Patients with significant medical illnesses, history of gastric surgery, and H. pylori eradication or use of antimicrobials or gastrointestinal medications, such as proton pump inhibitors or bismuth compounds in the past 2 months, were excluded from the study. The study was performed according to the good clinical practices and the Declaration of Helsinki guidelines. The study protocol was approved by the Ethics Committee for Research Involving Human Subjects (EC-58-58). Written informed consent was obtained from the participants.
Gastric tissue specimens
The EGD procedures were performed using an upper gastrointestinal video endoscope (Olympus EVIS EXERA III, CV-190, Japan). The whole stomach was examined and biopsied using a site-specific biopsy technique to analyze these specimens for histopathology (18).
Diagnosis of H. pylori infection
H. pylori-associated gastritis was observed during histopathological examination. Biopsy samples were obtained directly from the observation area, and H. pylori was detected using a rapid urease test on site (ProntodyleR, GASTREX, France). H. pylori infection was proven by polymerase chain reaction (PCR).
DNA preparation
A total of 400 gastric formalin-fixed paraffin-embedded (FFPE) tissues were used for genomic DNA extraction using the QIAamp DNA FFPE Tissue Kit (Qiagen, Dusseldorf, Germany). The process of genomic DNA extraction was performed according to the manufacturer’s instructions. Briefly, the paraffin-embedded tissues were deparaffinized in xylene using three changes for 5 min each and hydrated in 100% ethanol and 95% ethanol. Samples were then subsequently extracted for genomic DNA by digested lysis buffer and proteinase K. The tissue lysate was purified using the QIAamp spin column and eluted and stored at −20 °C.
Genotyping of TLR gene polymorphisms
Three single-nucleotide polymorphisms (SNPs) were selected according to the National Center for Biotechnology Information SNP database. TLR2 SNP rs3804099 (T>C) and rs3804100 (T>C) and TLR4 SNP rs10759932 (T>C) were used in the present study. TLR(s) polymorphism was determined by TaqMan allelic discrimination with a TaqMan SNP Genotyping Assay using a real-time PCR. Forward and reverse primers were designed by the wild-type probe VIC and the variant probe FAM. Real-time PCR was performed according to the manufacturer’s instructions (LightCycler® 480 II instrument Roche diagnostics, Neuilly-sur-Seine, France). Briefly, the PCR conditions were as follows: 95 °C for 10 min, 55 cycles of 95 °C for 15 s, and 60 °C for 1 min. The genotypes of polymorphisms were analyzed by the LightCycler® 480 software 1.5 (Roche Diagnostics, Neuilly-sur-Seine, France). The genotyping success rate for each SNP was >94%.
Statistical analysis
All statistical analyses were performed by the Statistical Package for the Social Sciences (SPSS) for Windows, version 20.0 (IBM Corp.; Armonk, NY, USA). The association among genotypes and the risk of gastric lesion development and H. pylori susceptibility were evaluated by the odds ratios (ORs) and 95% confidence intervals (95% CIs) from logistic regression analyses. A p<0.05 was considered as statistically significant.
RESULTS
Patient characteristics and TLR genotyping in clinical samples
A total of 400 patients (136 men and 264 women) with gastritis participated in the study. The median age of the patients was 44.6±15.9 years. Of these patients, 204 cases were infected with H. pylori, and 196 were H. pylori negative. Gastric lesions included chronic gastritis (n=312), gastric atrophy (GA) (n=26), internal metaplasia (IM) (n=38), and GC (n=24). SNPs of TLR2 rs3804099 and rs3804100 and TLR4 rs10759932 were genotyped in all patients. The proportions of T/T, T/C, and C/C genotypes were 64%, 17.25%, and 18.75% for TLR2 rs3804099; 74%, 23%, and 3% for TLR2 rs3804100; and 73%, 22.5%, and 4.5% for TLR4 rs10759932, respectively. Table 1 shows the baseline characteristics of the subjects.
Table 1.
Summary of subject characteristics
| Characteristics | Total=400 (%) |
|---|---|
| Male/female (N) | 136/264 |
| Median age±SD (years) | 44.6±15.9 |
| H. pylori infection | |
| Negative | 196 (49) |
| Positive | 204 (51) |
| TLR2 rs3804099 | |
| TT | 256 (64) |
| TC | 69 (17.25) |
| CC | 75 (18.75) |
| TLR2 rs3804100 | |
| TT | 296 (74) |
| TC | 92 (23) |
| CC | 12 (3) |
| TLR4 rs10759932 | |
| TT | 292 (73) |
| TC | 90 (22.5) |
| CC | 18 (4.5) |
| Gastric mucosal morphological patterns | |
| Chronic gastritis | 312 (78) |
| Internal metaplasia | 38 (9.5) |
| Gastric atrophy | 26 (6.5) |
| Gastric cancer | 24 (6) |
Association of TLR polymorphism with gastric lesions
For determination of the association between genotype and gastric lesions, a total of 400 patients were divided into two groups: 312 patients of the non-malignant (chronic gastritis) group and 88 patients of the premalignant (IM and GA) and/or malignant (GC) group. As shown in Table 2, TLR4 rs10759932, C/C homozygous genotype was associated with premalignant/malignant and had a significantly increased risk of premalignant/malignant development (OR=2.48, 95% CI=1.96–4.62, p=0.015) (Table 2).
Table 2.
Association between TLR genotype and gastric lesions
| Polymorphism | Gastric lesions | p* | ||
|---|---|---|---|---|
|
| ||||
| Non-malignant | Premalignant (GA/IM)/malignant (GC) | OR (95% CI) | ||
| TLR2 rs3804099 | ||||
| TT | 194 (62.18) | 62 (70.46) | 0.73 (0.61–0.87) | 0.192 |
| TC | 56 (17.95) | 13 (14.77) | 0.82 (0.57–0.95) | 0.261 |
| CC | 62 (19.87) | 13 (14.77) | 0.61 (0.38–0.77) | 0.183 |
| TLR2 rs3804100 | ||||
| TT | 230 (73.72) | 66 (75) | 0.78 (0.62–0.91) | 0.838 |
| TC | 70 (22.43) | 22 (25) | 0.69 (0.43–0.87) | 0.746 |
| CC | 12 (3.85) | 0 | - | - |
| TLR4 rs10759932 | ||||
| TT | 216 (69.23) | 76 (86.36) | 0.75 (0.59–0.83) | 0.471 |
| TC | 90 (28.85) | 0 | - | - |
| CC | 6 (1.92) | 12 (13.64) | 2.48 (1.96–4.62) | 0.015* |
GA: gastric atrophy; IM: intestinal metaplasia; GC: gastric cancer
Significance is set at p<0.05
Association of TLR polymorphism with H. pylori susceptibility
The association between TLR polymorphism and H. pylori infection was divided by genotypes (T/T, T/C, and C/C), dominant model (T/T vs. T/C+C/C), and recessive model (T/T+C/C vs. C/C). A significant association was observed between the recessive model of TLR4 rs10759932 and H. pylori status. The recessive model showed a decreased risk of H. pylori susceptibility (adjusted OR=0.52, 95% CI=0.38–0.82, p=0.046) (Table 3).
Table 3.
Association between recessive genotype and H. pylori infection
| Polymorphism | H. pylori status | OR (95% CI) | p | |
|---|---|---|---|---|
|
| ||||
| Negative | Positive | |||
| TLR2 rs3804099 | ||||
| TT | 131 (66.84) | 125 (61.27) | 1 | 0.721 |
| TC | 41 (20.92) | 28 (13.73) | 0.96 (0.62–1.29) | 0.543 |
| CC | 24 (12.24) | 51 (25) | 0.86 (0.58–0.97) | 0.261 |
| Dominant | ||||
| TT | 131 (66.84) | 125 (61.27) | 1 | 0.462 |
| TC+CC | 65 (33.16) | 79 (38.73) | 0.68 (0.59–0.91) | 0.158 |
| Recessive | ||||
| TT+TC | 172 (87.76) | 153 (75) | 1 | 0.663 |
| CC | 24 (12.24) | 51 (25) | 0.78 (052–0.91) | 0.328 |
| TLR2 rs3804100 | ||||
| TT | 149 (76.02) | 147 (72.06) | 1 | 0.843 |
| TC | 43 (21.94) | 49 (24.02) | 0.98 (0.83–1.25) | 0.924 |
| CC | 4 (2.04) | 8 (3.92) | 0.74 (0.53–0.93) | 0.548 |
| Dominant | ||||
| TT | 149 (76.02) | 147 (72.06) | 1 | 0.614 |
| TC+CC | 47 (23.98) | 57 (27.94) | 0.79 (0.56–0.97) | 0.627 |
| Recessive | ||||
| TC+CC | 47 (23.98) | 57 (27.94) | 1 | 0.283 |
| CC | 4 (2.04) | 8 (3.92) | 0.84 (0.63–0.97) | 0.365 |
| TLR4 rs10759932 | ||||
| TT | 139 (70.92) | 153 (75) | 1 | 0.442 |
| TC | 53 (27.04) | 37 (18.14) | 0.58 (0.43–0.82) | 0.624 |
| CC | 4 (2.04) | 14 (6.86) | 0.62 (0.43–0.92) | 0.068 |
| Dominant | ||||
| TT | 139 (70.92) | 153 (75) | 1 | 0.441 |
| TC+CC | 57 (29.08) | 51 (25) | 0.87 (0.63–0.98) | 0.472 |
| Recessive | ||||
| TT+TC | 192 (97.96) | 190 (93.14) | 1 | 0.793 |
| CC | 4 (2.04) | 14 (6.86) | 0.52 (0.38–0.82) | 0.046* |
Significance is set at p<0.05
Association of H. pylori infection with risk of gastric lesions by recessive model
We further evaluated the association between H. pylori infection and risk of gastric lesions in subpopulations stratified by the recessive model. Table 4 shows the results. In subjects without H. pylori infection, the recessive model was associated with an increased risk of non-malignant or chronic gastritis (OR=3.46, 95% CI=2.25–5.67, p=0.001). Meanwhile, in subjects with H. pylori infection, the recessive model was associated with an increased risk of both non-malignant (OR=2.28, 95% CI=1.24–3.57, p=0.001) and premalignant/malignant (OR=1.83, 95% CI=1.16–2.84, p=0.027).
Table 4.
Risk of gastric lesions associated with combined effect of recessive model and H. pylori infection
| Gastric mucosal pathology | H. pylori | TLR4 rs10759932 | OR (95% CI) | p | |
|---|---|---|---|---|---|
|
| |||||
| TT+TC | CC | ||||
| Non-malignant | − | 186 (59.62) | 5 (1.60) | 3.46 (2.25–5.67) | 0.001* |
| + | 116 (37.18) | 5 (1.60) | 2.28 (1.24–3.57) | 0.001* | |
| Pre-/malignant | − | 17 (17.71) | 0 | - | - |
| + | 68 (70.83) | 11 (11.46) | 1.83 (1.16–2.84) | 0.027* | |
Significance is set at p<0.05
DISCUSSION
To our knowledge, this is the first study to assess the SNPs of TLR2 rs3804099 and rs3804100 and TLR4 rs10759932 in association with risk of gastric lesion development and H. pylori susceptibility. In the present study, we found that TLR4 rs10759932, but not TLR2 rs3804099 and rs3804100, was associated with risk of premalignant/malignant and H. pylori susceptibility. TLR4 rs10759932, C/C homozygous genotype had an increased risk of premalignant gastric lesions. The C/C homozygous might change the expression level or structure of the TLRs that modify the signaling pathway. Meanwhile, the recessive model was associated with a reduced risk of H. pylori susceptibility. These may have a protective effect on host susceptibility to H. pylori infection, or functional impairment of TLR4 is crucial during H. pylori-induced gastric diseases.
We further tested the combined effects between the recessive model of TLR4 rs10759932 and H. pylori infection on risk of gastric lesions. The results showed a significant association with an increased risk of non-malignant and premalignant/malignant in patients with or without H. pylori infection. H. pylori infection appears to contribute to chronic gastritis, and premalignant/malignant supported the development of GC involved in H. pylori infection. However, no multiplicative or additive interaction of SNP H. pylori was addressed. Additionally, the results of H. pylori positive patients revealed an approximately 1.25-fold reduced risk of premalignant compared with non-malignant. Thus, these analyses suggested that there was an interaction between the recessive model TLR4 rs10759932 and H. pylori infection.
It has been shown that TLR polymorphisms can influence H. pylori susceptibility. H. pylori-related diseases developed during modulation of pro-inflammatory or anti-inflammatory cytokines. Recent genetic association studies have suggested that chronic inflammation is a risk factor associated with the development of GC (19). TLR genotypes might alter cytokine production resulting in modulation of H. pylori susceptibility, chronic inflammation, and GC (20,21). TLR2 polymorphisms are associated with susceptibility to GC in various ethnic groups, such as Japanese, Chinese, and Caucasian population. TLR4 polymorphisms have also been associated with gastritis and GC in Europe, Asia, and America (22,23). In an ethnic Kashmiri population, a previous study observed a relationship between TLR4 polymorphisms and gastric diseases (24), and a weak association was seen in the Japanese population (25,26). Similar to other infectious pathogens, genetic polymorphism in the TLR4 gene reduces inflammation, causing less damage and persistent infection (27–29). However, TLR4 mRNA was up-regulated in H. pylori-infected gastric epithelial cell lines (30).
Thai patients might explain the typically benign outcome of H. pylori-related diseases. Our study provides additional evidence to further support the hypothesis that the outcome of the infection is largely influenced by the immune response or infectious susceptibility of the host. H. pylori-related host genetic factors independent of or combined with H. pylori infection appear to play important roles in modulating the risk of the development of gastric carcinogenesis. However, multicenter studies are needed for larger studies to test this hypothesis. The role of TLR genetic polymorphisms of GC should be extensively evaluated by studying the distribution of TLR polymorphisms in the Thai population. Further studies of TLR expression and H. pylori colonization with the assessment of the cytokine responses are needed to confirm the impact of downstream signaling on GC susceptibility.
Footnotes
Ethics Committee Approval: Ethics committee approval was received for this study from the ethics committee of Suranaree University of Technology (Decision Date: 01.12.2015; Decision No: EC-58-59).
Informed Consent: Written informed consent was obtained from the patients who participated in this study.
Author Contributions: Concept - T.T., C.D.; Design - T.T. T.S. Supervision - T.T., T.S., C.D., W.L.; Resources - T.T, T.S.; Materials - T.T., T.S.; Data Collection and/or Processing - T.S., T.T.; Analysis and/or Interpretation - T.S., T.T.; Literature Search - T.T., T.S., W.W.; Writing Manuscript - T.T., T.S., W.W.; Critical Reviews - T.T., T.S.
Conflict of Interest: The authors have no conflict of interest to declare.
Financial Disclosure: This research was supported by a grant from Suranaree University of Technology and by office of the higher education commission under NRU project of Thailand.
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