Effect of the −2081G/A Polymorphism of the TLR4 Gene and Its Interaction with Helicobacter pylori Infection on the Risk of Gastric Cancer in Chinese Individuals

Abstract

Aims: Toll-like receptor 4 (TLR4) plays an important role in gastric carcinoma. Using a case-control study, we analyzed the genotypic distribution of TLR4 rs10983755 (−2081G/A) and rs11536878 in a Chinese population and investigated the effect of their interactions with Helicobacter pylori infection on susceptibility to gastric cancer (GC) and atrophic gastritis (AG). Methods: In this study, 409 and 581 cases of GC and AG, respectively, were selected for analyses along with an equal number of matched controls. The TLR4 polymorphisms were genotyped using Sequenom MassARRAY. Serum levels of anti-H. pylori IgG were determined by enzyme-linked immunosorbent assay. Results: The TLR4-2081G/A polymorphism was negatively associated with GC (AG+AA vs. GG: odds ratio [OR]=0.70, 95% confidence interval [CI]=0.53-0.93, p=0.012). A decreased risk of GC was observed in H. pylori negative and TLR4-2081(AG+AA) genotype subjects [H. pylori(−)/AG+AA vs. H. pylori(+)/GG: OR=0.16, 95% CI=0.09-0.27, p<0.001]. The rs11536878 polymorphism was not associated with GC or AG. Conclusions: The TLR4-2081G/A polymorphism seems to affect the risk of gastric carcinogenesis and may to some degree play a protective role against H. pylori infection.

Background

Gastric cancer (GC) is a malignancy with high incidence and mortality rates worldwide. According to statistics, 989,600 new cases of GC and 738,000 related deaths occurred in 2008 worldwide, accounting for 8% of all new cancer cases and 10% of cancer deaths, respectively (Jemal et al., 2010). Notably, 70% of new cases and deaths occurred in developing countries. The high regional incidence of GC is partly due to Helicobacter pylori infections. In 1994, H. pylori was classified as a category I carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) (1994). H. pylori plays a crucial role in the progression from chronic atrophic gastritis (AG) to gastric atrophy, metaplasia, atypical hyperplasia, and ultimately intestinal-type GC (Correa and Piazuelo, 2012). However, when exposed to the same levels of H. pylori infection, some individuals remain uninfected, whereas the familial aggregation of GC occurs in other individuals (Garcia-Gonzalez et al., 2007; Demirel et al., 2013). Recent studies have shown that host genetic factors (particularly polymorphisms in certain immune and inflammation-related genes) play essential roles in GC, in addition to environmental factors (Garcia-Gonzalez et al., 2012; Xue et al., 2012).

The toll-like receptor (TLR) family is an important class of pattern recognition receptors (PRRs) in humans that recognize pathogen-associated molecular patterns (Ishihara et al., 2004). TLR4 recognizes exogenous ligands such as lipopolysaccharides (LPS), which are major components of the H. pylori outer membrane. Therefore, TLR4 plays an important role in H. pylori recognition and colonization as well as H. pylori-induced gastrointestinal inflammation and gastric carcinogenesis (Chochi et al., 2008; Yokota et al., 2012). TLR4 gene polymorphisms might cause variations in host susceptibilities to the same infectious agent. For instance, the missense polymorphisms Asp299Gly and Thr399Ile in the coding region of the TLR4 gene alter the conformation of the extracellular ligand-binding domain of TLR4, resulting in a reduced ability of the host to respond to LPS (Hold et al., 2007). In addition, these two genetic polymorphisms are closely associated with susceptibility to GC and precancerous gastric diseases (Arbour et al., 2000; Garcia-Gonzalez et al., 2007). However, a number of studies conducted in China, Japan, and South Korea have revealed that genotypic frequencies of the Asp299Gly and Thr399Ile polymorphisms in TLR4 are nonexisting in Asian populations (Wu et al., 2006; Tahara et al., 2007).

In addition to Asp299Gly and Thr399Ile, other polymorphisms have been identified in the TLR4 gene. Studies have suggested that the rs10983755 polymorphism, located 2081 bp upstream of the TLR4 gene's transcription initiation site, may affect the binding of transcription factors to the gene promoter region (Ragnarsdottir et al., 2010). The rs11536878 polymorphism, located in the intron area of the TLR4 gene, has been reported to be associated with an increased risk of developing neutropenia (Miedema et al., 2011). However, the relationships between the rs10983755 and rs11536878 polymorphisms and GC (including precancerous lesions) are still not well understood. Therefore, in the present investigation, case-control studies were performed to determine the genotypic distribution of the TLR4 rs10983755 and rs11536878 polymorphisms in a Chinese population and to evaluate the effects of the interactions between these genotypes and H. pylori infection on the susceptibility to GC and AG.

Materials and Methods

Ethics statement

All research subjects signed informed consent documents, and our study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University.

Study population and data collection

A population-based case-control study was performed. In total, 1854 participants were enrolled in the study. The case group included 478 cases of GC and 652 cases of AG, whereas the control group included 724 cases of mild superficial gastritis. The subjects were either participants in a health check program for GC screening in the Zhuanghe district of Liaoning Province, China, or patients at the First Affiliated Hospital of China Medical University in Liaoning province enrolled between March 2002 and December 2011. All subjects in this study were endoscopically and histologically confirmed. Superficial gastritis and AG were diagnosed in accordance with the Sydney pathological diagnostic criteria (Dixon et al., 1996). Gender and age information was retrospectively extracted from registration documents and questionnaires.

For individual association analyses of TLR4 genotypic effects on GC and AG risk, cases and controls were matched at a ratio of 1:1 according to age (±5 years) and gender. Ultimately, 409 and 581 cases of GC and AG, respectively, were selected for analyses along with an equal number of matched controls.

Genotyping

Genomic DNA was extracted from plasma using the standard phenol-chloroform method, diluted to a final concentration of 50 ng/μL, and loaded randomly into 384-well microtiter plates. Polymorphisms were analyzed using the Sequenom MassARRAY platform (a matrix-assisted laser desorption ionization time-of-flight mass spectrometry platform [Sequenom, San Diego, CA]), as previously described by He et al. (2013). One hundred samples were randomly selected, and the genotyping was repeated using DNA sequencing. The data obtained in both methods were in complete agreement.

Examination of H. pylori serology

H. pylori IgG antibodies in serum samples were examined using the H. pylori IgG enzyme-linked immunosorbent assay (ELISA) kit (Biohit, Helsinki, Finland) according to the manufacturer's instructions. Antibody titers equal to or greater than 34 enzyme immunoassay units (EIU) were considered positive.

Statistical methods

Statistical analyses were performed using the SPSS 13.0 software (SPSS, Chicago, IL). HWE and gender differences between the various disease groups were compared using the Pearson's χ² test. Age differences between disease groups were evaluated by ANOVA. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to represent the risk of developing various gastric diseases. ORs were calculated using multivariate logistic regression analyses, after adjusting for age and gender. p-Values for the interactions between genotypes and H. pylori were calculated using likelihood ratio tests. All statistical tests were two-sided probability tests with a p-value less than 0.05 considered statistically significant.

Results

Characteristics of the study population

Basic information on all participants is summarized in Table 1. Significant differences in gender and age were observed between cases and controls in the total population. But because of the frequency matching used in our study design, there were no differences in gender or age between case and control groups in the matched samples for both variants, which were used for final analyses of individual genetic effects on GC and AG. However, in matched samples, cases were more likely to have H. pylori infection (p<0.001).

Table 1.

Baseline Characteristics of the Study Participants for TLR4 rs10983755

	Superficial gastritis vs. atrophic gastritis			Superficial gastritis vs. gastric cancer
Variable	Superficial gastritis	Atrophic gastritis	p-Value	Superficial gastritis	Gastric cancer	p-Value
Total sample (n)	724	652		724	478
Age (mean±SD)	53.07±9.84	55.02±8.97	<0.001	53.07±9.84	59.02±11.08	<0.001
Range	17-85	17-82		17-85	26-85
Gender	724	652		724	478	<0.001
Male	367 (50.7%)	369 (56.6%)		367 (50.7%)	326 (68.2%)
Female	357 (49.3%)	283 (43.4%)		357 (49.3%)	152 (31.8%)
Matched sample (n)	581	581		409	409
Age (mean±SD)	54.55±8.99	54.61±8.90	0.883	56.13±9.123	56.27±9.293	0.832
Range	17-85	17-82		26-85	26-84
Gender	581	581		409	409	1
Male	320 (55.1%)	320 (55.1%)		278 (32.0%)	278 (32.0%)
Female	261 (44.9%)	261 (44.9%)		131 (68.0%)	131 (68.0%)
Helicobacter pylori serology	575	574		404	215
Positive	117 (20.3)	358 (62.4)		87 (21.5)	111 (51.6)
Negative	458 (79.7)	216 (37.6)	<0.001	317 (78.5)	104 (48.4)	<0.001

TLR4, toll-like receptor 4.

Genotyping and its association with GC and AG risk

The frequency distributions of the rs10983755 and rs11536878 genotypes are summarized in Table 2. With regard to the rs11536878 polymorphism genetic frequencies, no significant differences between cases and controls were present, either in the GC group or in the AG group. In the AG group, subjects with rs10983755 AG or AA genotypes did not harbor a significantly different risk of AG compared with the common GG genotype. However, using rs10983755 GG genotype as a reference in the GC group (OR=1.00), ORs for AG and (AG+AA) genotypes were 0.67 (95% CI: 0.50-0.90; p=0.007) and 0.70 (95% CI: 0.53-0.93; p=0.012), respectively, showing that subjects with rs10983755 AG or (AG+AA) genotypes had a statistically significant decreased risk of GC.

Table 2.

Genotype Distribution OF TLR4 Among Matched Samples and Association with Gastric Cancer Risk

	Controls vs. atrophic gastritis				Controls vs. gastric cancer
Genotype	Controls, n (%)	Atrophic gastritis, n (%)	Adjusted OR (95% CI) ^a	p-Value	Controls, n (%)	Gastric cancer, n (%)	Adjusted OR (95% CI) ^a	p-Value
rs10983755	581	581			409	409
GG	294 (50.6)	290 (49.9)	1 (ref)		177 (43.3)	213 (52.1)	1 (ref)
AG	234 (40.3)	243 (41.8)	1.05 (0.83-1.34)	0.676	188 (46.0)	152 (37.2)	0.67 (0.50-0.90)	0.007^b
AA	53 (9.1)	48 (8.3)	0.92 (0.60-1.40)	0.695	44 (10.8)	44 (10.8)	0.83 (0.52-1.33)	0.445
AG+GG	287 (49.4)	291 (50.1)	1.03 (0.82-1.29)	0.815	232 (56.7)	196 (47.9)	0.70 (0.53-0.93)	0.012^b
rs11536878	581	581			409	409
CC	467 (80.4)	461 (79.3)	1 (ref)		328 (80.2)	331 (80.9)	1 (ref)
CA	106 (18.2)	115 (19.8)	1.09 (0.82-1.47)	0.528	77 (18.8)	74 (18.1)	0.95 (0.67-1.35)	0.773
AA	8 (1.4)	5 (0.9)	0.63 (0.21-1.95)	0.426	4 (1.0)	4 (1.0)	0.99 (0.25-3.98)	0.986
CA+AA	114 (19.6)	120 (20.7)	1.07 (0.80-1.42)	0.661	81 (19.8)	78 (19.1)	0.95 (0.67-1.35)	0.777

ORs were calculated by logistic regression and adjusted for age and gender.

Statistical significance at p<0.05 with a two-sided test.

OR, odds ratio; 95% CI, 95% confidence interval.

We further evaluated the association between TLR4 genotypes and the risk of GC and AG by gender and age. No significant association was found between the rs11536878 genotype and either AG or GC, as shown in Table 4. However, a decreased risk of GC associated with the rs10983755 (AG+AA) genotype was pronounced in individuals aged ≤60 years (adjusted OR=0.68, 95% CI: 0.49-0.96; p=0.027) and in males (adjusted OR=0.68, 95% CI: 0.48-0.95; p=0.022), as shown in Table 3.

Table 3.

Stratification Analysis of Association Between TLR4 rs11536878 Genotype and Gastric Cancer Risk

Variation	Genotype	Controls vs. atrophic gastritis	Adjusted OR (95% CI) ^a	p-Value	Controls vs. gastric cancer	Adjusted OR (95% CI) ^a	p-Value
Age
≤60	CC	343/338	1 (ref)		223/230	1 (ref)
	CA+AA	81/89	1.12 (0.79-1.56)	0.526	51/46	0.87 (0.56-1.35)	0.540
>60	CC	124/123	1 (ref)		105/101	1 (ref)
	CA+AA	33/31	0.95 (0.55-1.64)	0.846	30/32	1.11 (0.63-1.96)	0.719
Gender
Male	CC	253/256	1 (ref)		218/230	1 (ref)
	CA+AA	67/64	0.94 (0.64-1.39)	0.769	60/48	0.76 (0.50-1.16)	0.197
Female	CC	214/205	1 (ref)		110/101	1 (ref)
	CA+AA	47/56	1.24 (0.81-1.92)	0.323	21/30	1.54 (0.83-2.88)	0.168

ORs were calculated by logistic regression and adjusted for age and gender.

Table 4.

Stratification Analysis of Association Between TLR4 rs10983755 Genotype and Gastric Cancer Risk

Variation	Genotype	Controls vs. atrophic gastritis	Adjusted OR (95% CI) ^a	p-Value	Controls vs. gastric cancer	Adjusted OR (95% CI) ^a	p-Value
Age
≤60	GG	219/210	1 (ref)		118/145	1 (ref)
	AG+AA	205/217	1.10 (0.84-1.44)	0.483	156/131	0.68 (0.49-0.96)	0.027^b
>60	GG	75/80	1 (ref)		59/68	1 (ref)
	AG+AA	82/74	0.85 (0.54-1.33)	0.471	76/65	0.73 (0.45-1.19)	0.208
Gender
Male	GG	147/156	1 (ref)		119/146	1 (ref)
	AG+AA	173/164	0.89 (0.65-1.22)	0.892	159/132	0.68 (0.48-0.95)	0.022^b
Female	GG	147/134	1 (ref)		58/67	1 (ref)
	AG+AA	114/127	1.22 (0.87-1.73)	0.254	73/64	0.76 (0.47-1.24)	0.271

ORs were calculated by logistic regression and adjusted for age and gender.

Statistical significance at p<0.05 with a two-sided test.

Effect of interaction between the rs10983755 genotype and H. pylori infection on the risk of GC and AG

In the present study, as shown in Table 5, an interactive analysis was further conducted to examine the effects of H. pylori infection on the relationship between the rs10983755 genotype and the risk of GC and AG. Using the GG genotype and H. pylori IgG (+) as the reference, the ORs for the (AG+AA) genotype and H. pylori(−) were 0.14 (95% CI: 0.09, 0.21) in the AG subgroup and 0.16 (95% CI: 0.09, 0.27) in the GC subgroup.

Table 5.

Interaction Between TLR4 rs10983755 Genotype and H. pylori Infection

	Controls vs. atrophic gastritis			Controls vs. gastric cancer
Variation	H. pylori(+)	H. pylori(−)	p-Value	H. pylori(+)	H. pylori(−)	p-Value
rs10983755
GG
Atrophic gastritis vs. controls (n)	51/179	240/107		30/63	144/47
Adjusted OR (95% CI)^a	1 (ref)	0.13 (0.86-0.19)		1 (ref)	0.16 (0.09-0.27)
AG+AA
Gastric cancer vs. controls (n)	66/179	218/109		57/48	173/57
Adjusted OR (95% CI)^a	0.77 (0.51-1.18)	0.14 (0.09-0.21)	<0.001^b	0.41 (0.23-0.73)	0.16 (0.09-0.27)	<0.001^b

ORs were calculated by logistic regression and adjusted for age and gender of cancer subgroups.

Statistical significance at p<0.05 with a two-sided test.

Discussion

H. pylori is an important pathogenic factor for GC. Although the specific mechanism of H. pylori-induced carcinogenesis is not fully understood, the theory proposed by Correa et al. that inflammation acts as a triggering factor for gastric carcinogenesis draws increasing attention (Correa et al., 2007). Indeed, the role of inflammation in H. pylori-induced gastric carcinogenesis has been confirmed (Correa and Houghton, 2007; Piazuelo et al., 2010). TLR4 protein is a type I transmembrane, PRR. In stomach tissues, TLR4 is mainly expressed in the epithelial cells of the gastric mucosa and in interstitial lymphocytes. Interestingly, TLR4 expression has been shown to be increased in gastric mucosal epithelial cells colonized by H. pylori (Schmausser et al., 2004; Yokota et al., 2010). Upon H. pylori infection of the gastric mucosa, the LPS-CD4 protein complex on the cell membrane interacts with the extracellular ligand-binding domain of TLR4. This results in the activation of the TLR4 signaling pathway, which causes the release of a large number of inflammatory factors (such as IL-1) and anti-inflammatory factors (such as IL-10) (Obonyo et al., 2007; Lu et al., 2012). It is thought that the long-term imbalance of inflammatory and anti-inflammatory factors might cause chronic injury in the gastric mucosa, resulting in H. pylori-related chronic inflammation of the gastric mucosa, precancerous disease, and even the development of GC (Ishihara et al., 2004; Pathak et al., 2006; Chochi et al., 2008).

Given that proteins are products of gene expression and subject to gene regulation, TLR4 gene polymorphisms may alter the function of the TLR4 protein, causing the body to lose its ability to handle pathogenic molecules (such as H. pylori LPS) and eventually long-term inflammatory injury in the gastric mucosa. Studies have shown that TLR4 protein expression is reduced in C3H/Hej mice (mutant mice with proline residue at position 712 of TLR4 replaced by histidine) (Poltorak et al., 1998). In addition, Asp299Gly and Thr399Ile polymorphisms alter the conformation of TLR4. Under these conditions, host cells are unable to respond to LPS and the LPS signaling pathway is inhibited (Arbour et al., 2000). Trejo-de la et al. reported that Asp299Gly and Thr399Ile induce changes in the secretion of mucosal inflammatory factors and anti-inflammatory factors in gastrointestinal diseases (Trejo-de la et al., 2008). Recent studies have suggested that Asp299Gly and Thr399Ile are genetic polymorphisms closely related to the development of GC and precancerous diseases. Although there is not a frequent distribution of these polymorphisms in Chinese populations, these findings indicated that gastric mucosal inflammation induced by TLR4 gene polymorphisms might be one of the mechanisms underlying the high incidence of H. pylori-induced GC in specific groups.

In addition to Asp299Gly and Thr399Ile polymorphisms in the TLR4 coding region, other polymorphisms occurring in the promoter region of the TLR4 gene have been described, including rs10983755, which is located at the position −2081. However, only a few studies have shown that rs10983755 polymorphism is associated with inflammatory diseases. Interestingly, recent studies have demonstrated that the A variant of rs10983755 is related to asymptomatic urinary tract infections in Swedish populations and development of nephritis and other kidney diseases in Taiwanese populations (Chena et al., 2010; Ragnarsdottir et al., 2010). In addition, Zhang et al. (2011) reported an association between the variant of rs10983755 and the severity of asthma attacks in Chinese populations. In the present study, we are the first to find that the distribution ratio of the rs10983755 polymorphism (AG+AA) genotype is significantly lower in GC cases than that of the GG genotype, indicating that the risk of GC in carriers of the (AG+AA) genotype of rs10983755 is lower than that in carriers of the GG genotype in the Han population of northern China. These findings suggest that, similar to Asp299Gly and Thr399Ile polymorphisms, the rs10983755 polymorphism might induce changes in immune-mediated inflammatory processes. Notably, analysis by gender and age revealed that correlation between polymorphisms and GC occurred in the male (rather than female) subgroup. Studies of brain injury have revealed that progesterone may affect TLR4 gene expression under certain conditions (De Nicola et al., 2013). Whether hormones cooperate with the TLR4 rs10983755 polymorphism to affect gastric carcinogenesis remains to be verified.

To further clarify the relationships between the TLR4 rs10983755 polymorphism and H. pylori infection in gastric carcinogenesis, we investigated the interaction between rs10983755 genotype and H. pylori in GC and its precancerous conditions. Remarkably, compared with the GG genotype, the (AG+AA) genotype did not independently reduce the risk of AG. However, in the context of H. pylori infection, the changes in risk of AG and GC became statistically significant. These important findings suggest that H. pylori plays a crucial role in the progression from chronic AG to gastric atrophy, and ultimately to GC as previously suggested (Correa and Piazuelo, 2012). In the progression of GC, H. pylori and the TLR4 gene play interactional roles, and the carriers of the A allele of TLR4 rs10983755 may exhibit increased resistance to H. pylori infections.

In this study, we did not find any association between the rs11536878 polymorphism and the risk of GC and AG. This may suggest that the rs11536878 polymorphism is not a functional polymorphism in the progression of GC or that the number of subjects in this study's sample was not large enough to detect the association.

When considering the results of the present study, the following limitations should be considered. First, the specific functions of the TLR4 rs10983755 polymorphism required further clarification by, for instance, identifying transcription factors related to the polymorphism and the changes in the inflammatory factors downstream of the TLR4 pathway. Second, while this study employed serological ELISA to detect the H. pylori infection, this assay does not provide perfect discrimination (i.e., 100% accuracy). However, it is a generally effective detection method used in China, Japan, and other countries with high rates of H. pylori infection (Sung et al., 2001; Goh et al., 2007; Toyoda et al., 2012). Finally, a portion of the samples used in this study was collected in advance and stored in a DNA biobank. As a result, not all samples contained enough serum to detect the rate of H. pylori infections (Sung et al., 2001; Goh et al., 2007; Toyoda et al., 2012). Given these limitations, future studies using larger samples should be conducted to confirm our findings and examine the potential mechanisms behind these associations.

In summary, the present investigation has demonstrated that, compared with the GG genotype, the (AG+GG) genotype of the TLR4 rs10983755 polymorphism seems to confer a decreased risk of developing GC in Chinese populations. In addition, the A allele of TLR4 rs10983755 displayed a certain protective effect against the damages induced by cancer-promoting H. pylori infections.

Footnotes

Acknowledgments

This study was supported by the National Basic Research Development Program of China (973 Program No. 2010CB529304), Funds for Scientific Research from the Financial Department of Liaoning Province (No. 2008-621), and the Science Technology Project in Liaoning Province (No.2011225002).

Author Contributions

Ping Li and Caiyun He performed the statistical analysis and wrote the article, contributing equally to these tasks. Qian Xu and Liping Sun were responsible for the collection of serum/biopsy samples, clinical information, and serological testing. Minwen Ha was responsible for collecting clinical information and revising the article. Yuan Yuan conceived and designed this study and revised the article.

Author Disclosure Statement

All authors declare that they have no financial conflicts of interest.

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