|Year : 2020 | Volume
| Issue : 1 | Page : 94-100
Association of toll-like receptors 2, 4, 9 and 10 genes polymorphisms and Helicobacter pylori-related gastric diseases in Saudi patients
Emad M Eed1, Yousry A Hawash2, Amany S Khalifa3, Khalaf F Alsharif4, Saleh A Alghamdi4, Abdulraheem A Almalki4, Mazen M Almehmadi4, Khadiga A Ismail5, Azza A Taha6, Taisir Saber7
1 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taif University, Taif, Saudi Arabia; Department of Medical Microbiology and Immunology, Faculty of Medicine, Menoufia University, Menoufia, Cairo, Egypt
2 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taif University, Taif, Saudi Arabia; Department of Molecular and Clinical Parasitology, National Liver Institute, Menoufia University, Menoufia, Cairo, Egypt
3 Department of Medical Microbiology and Immunology, Faculty of Pharmacy, Taif University, Taif, Saudi Arabia; Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Cairo, Egypt
4 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taif University, Taif, Saudi Arabia
5 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taif University, Taif, Saudi Arabia; Department of Parasitology, Faculty of Medicine, Ain-Shams University, Cairo, Egypt
6 Department of Public Health and Community Medicine, Faculty of Medicine, Menoufia University, Menoufia, Cairo, Egypt
7 Department of Medical Microbiology and Immunology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
|Date of Submission||18-Apr-2020|
|Date of Decision||23-May-2020|
|Date of Acceptance||03-Jun-2020|
|Date of Web Publication||25-Jul-2020|
Dr. Emad M Eed
College of Applied Medical Sciences, King Abdulaziz Campus, Taif
Source of Support: None, Conflict of Interest: None
Purpose: Helicobacter pylori is one of the most prevalent human pathogens worldwide. However, the outcomes of H. pylori infection are markedly variable from asymptomatic mild lesion to malignant transformation. Many factors are suggested to influence these infection outcomes, including host immunity and genetic susceptibility. Toll-like receptors (TLRs) can recognise different microbial components and play an essential role in the mucosal immune response against H. pylori infection. Materials and Methods: The association between the common single nucleotide polymorphisms (SNPs) in the genes of TLR2, 4, 9 and 10 and H. pylori-related gastric diseases were investigated by molecular methods after the confirmation of H. pylori infection. The study included 210 patients in three groups; chronic gastritis (n = 90), peptic ulcer disease (PUD) (n = 75) and gastric carcinoma (n = 45). Results: The results showed a significant association between TLR4 SNPs (rs 4986790 and rs 4986791) and the presence of H. pylori infection, especially in chronic gastritis patient group. Furthermore, TLR9-rs352140 TT genotype was more prevalent among chronic gastritis patient group. TLR10-rs 10004195 TT genotype was found to be less prevalent among H. pylori-related chronic gastritis and PUD and was suspected to have a protective effect. TLR2 SNPs (rs3804099 and rs3804100) showed no significant statistical difference between H. pylori-infected patients and the controls. Conclusion: TLR genes polymorphisms may play a role in H. pylori infection susceptibility and may influence its outcomes; however, the ethnic and other factors may modify this effect.
Keywords: Genes, Helicobacter pylori, polymorphisms, Saudi patients, toll-like receptors
|How to cite this article:|
Eed EM, Hawash YA, Khalifa AS, Alsharif KF, Alghamdi SA, Almalki AA, Almehmadi MM, Ismail KA, Taha AA, Saber T. Association of toll-like receptors 2, 4, 9 and 10 genes polymorphisms and Helicobacter pylori-related gastric diseases in Saudi patients. Indian J Med Microbiol 2020;38:94-100
|How to cite this URL:|
Eed EM, Hawash YA, Khalifa AS, Alsharif KF, Alghamdi SA, Almalki AA, Almehmadi MM, Ismail KA, Taha AA, Saber T. Association of toll-like receptors 2, 4, 9 and 10 genes polymorphisms and Helicobacter pylori-related gastric diseases in Saudi patients. Indian J Med Microbiol [serial online] 2020 [cited 2021 Jan 16];38:94-100. Available from: https://www.ijmm.org/text.asp?2020/38/1/94/290687
| ~ Introduction|| |
Helicobacter pylori is a Gram-negative spiral microaerophilic bacterium that widely (up to 50%) colonises the human gastric epithelium worldwide. It is recognised as a major aetiology of many pathological conditions, including gastritis, peptic ulcer, gastric carcinoma (GC) and mucosa-associated lymphoid tissue lymphoma. Besides its unique ability to overcome the gastric acidity, its high motility and its ability of cytotoxins production, H. pylori possess many factors that appear to reduce the inflammation and evade the host immune response. In addition to the bacterial and the environmental factors, host genetic polymorphisms related to the innate and adaptive immune systems have been proposed as possible predictors of H. pylori infection clinical outcomes.H. pylori infection is followed by neutrophils and mononuclear cells activation with the production of both pro-inflammatory and anti-inflammatory cytokines. Polymorphisms among genes that influence immune cell activation and/or cytokine production may cause disequilibrium between aggressive and protective factors acting in the host's mucosa and hence influence the individual's clinical outcome. Toll-like receptors (TLRs) belong to a large family called pattern recognition receptors (PRRs). In humans, ten TLRs have been well identified on the surfaces of immune-competent cells as well as other cells, including gastric epithelial cells., TLRs are expressed in Type I transmembrane proteins pattern, and their recognition results in activation of nuclear factor-kappa β (NF-κβ), induction of cytokines production and expression of antigen-presenting molecules. These signalling cascades induce several co-stimulatory molecules essential for adaptive immune responses. TLRs can recognise different microbial components; surface components as lipopolysaccharides (LPS) and lipoproteins could be recognised by TLR1, 2, 4, 5 and 6, while microbial nucleic acids could be recognised by TLR3, 7, 8 and 9.,, Furthermore, TLR10 is expressed on the surface gastric epithelial cells and involved in recognition of multiple distinct patterns of H. pylori LPS. Previous studies have demonstrated the role of TLRs along with interleukins (ILs), chemokines and adhesion molecules in the mucosal immune response against H. pylori infection. The studies revealed that the gastric epithelial cells TLRs expression was higher in H. pylori-infected than non-infected gastric mucosa.,, Furthermore, in the late stages of H. pylori infection, the intense expressions of TLRs likely activate pro-oncogenes leading to epithelial dysplasia and adenocarcinoma. Although H. pylori infection constitutes a major risk factor for gastric malignancy, only 1%–2% of the H. pylori-infected individuals develop gastric cancer. H. pylori infection outcomes, including malignancy, are related to many bacterial and host genetic factors. A previous European study has revealed an association between TLRs genetic variation and H. pylori prevalence rate, indicating that these polymorphisms might play a role in modulation of the host immune response and the susceptibility to H. pylori infection. Furthermore, recent studies revealed a significant association between the presence of single-nucleotide polymorphisms (SNPs) of TLRs and gastric cancer development. These SNPs might enable H. pylori for a prolonged infection, subsequently, may lead to chronic inflammation and carcinogenesis.,, However, the association between SNPs of TLRs and H. pylori infection's outcomes is still controversial among different ethnic populations. To the best of our knowledge, the impact of TLRs polymorphisms on the H. pylori-related gastric diseases has not yet been investigated in Saudi Arabia. Hence, the investigation of these polymorphisms may provide novel insights to improve the management of H. pylori-related gasteropathies among the Saudi population. The aim of this work was to investigate the association of the common SNPs in TLR2, 4, 9 and 10 with H. pylori-related gastric diseases among Saudi patients.
| ~ Materials and Methods|| |
Study design and population
This is a case-control study that was conducted during the period from April 2017 to September 2019. The study was approved by the Research Ethics Committee at Taif University. All participants were oriented about the study, and written consent was obtained from every patient. Two hundred and ninety patients were selected from the distinct public and private hospitals in the Taif region, Saudi Arabia. The patients were undergoing gastroscopy to investigate for upper gastrointestinal complaints related to H. pylori diseases (defined according to the Rome IV criteria). According to the endoscopic findings and histopathological diagnosis, patients were retrospectively categorised into three groups; Group 1 (90 patients) diagnosed with chronic gastritis, Group 2 (75 patients) diagnosed as peptic ulcer disease (PUD) and Group 3 (45 patients) diagnosed with GC. In addition to H. pylori-negative control group (80 patients). Data regarding medical history, physical examination and endoscopic findings were recorded. Patients with a recent history (within the last 4 weeks) of anti-H. pylori eradication therapy were excluded from the study.
Biopsy specimens and diagnosis of Helicobacter pylori
From each patient, two biopsies were collected from the gastric antrum and body. The biopsies were examined by a rapid urease test (RUT) (Pylori Tek, Serim Research, Elkhart, Indiana, US) to screen for H. pylori infection. Molecular confirmation to determine the final H. pylori positive status was done by GenoType HelicoDR testing (Hain Life Science GmbH, Nehren, Germany). H. pylori-negative biopsies (based on both RUT and GenoType HelicoDR test) from 80 patients without significant endoscopic findings were also collected as controls.
Genomic DNA was extracted from the gastric formalin-fixed paraffin-embedded (FFPE) biopsies using QIAamp DNA FFPE Tissue Kit (Qiagen, Santa Clara, CA) according to the manufacturer's instructions. The extracted DNA was stored at −20°C for genotyping assays of the six common SNPs in TLR2, TLR4, TLR9 and TLR10 genes that had been reported with functional effects or association with H. pylori infection outcome.,,,,
Genotyping of toll-like receptors gene polymorphisms
TLR2 (rs3804099 and rs3804100) and TLR10-rs10004195 SNPs were determined by TaqMan allelic discrimination genotyping assay (Applied Biosystems, Waltham, Massachusetts, US). Real-time polymerase chain reaction (PCR) was performed according to the manufacturer's instructions by Light Cycler 480 II instrument (Roche Diagnostics, Indianapolis, Indiana, US) as described before. Briefly, the PCR conditions were as follows: the initial cycle of 95°C for 10 min, followed by 55 cycles of 95°C for 15 s, and final extension at 60°C for 1 min. TLR4 (rs4986790 and rs4986791) and TLR9-rs352140 SNPs were investigated by restriction fragment length polymorphism (PCR-RFLP). TLR4 SNPs were determined as described by Michel et al. Briefly, initial cycle at 94°C for 10 min, 35 cycles at 94°C for 30 s, 61°C for 40 s (59°C in case of rs4986791), then final extension at 72°C for 10 min. PCR products were digested by restriction enzymes and then were electrophoresed in a 12% polyacrylamide gel. TLR9-rs352140 SNP was genotyped through PCR-RFLP as described by Loganathan et al. The primers sequences, restriction enzymes and restriction fragments of wild-type and mutant alleles are shown in [Table 1].
|Table 1: The primers sequences, restriction enzymes and fragment size of toll-like receptor 4 and toll-like receptor 9 polymerase chain reaction-restriction fragment length polymorphism analysis|
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Statistical analysis was performed using SPSS, version 22 (SPSS Inc., Chicago IL, USA), genotype frequencies were directly counted. The genotype distribution in H. pylori-infected patients and the controls were analysed using Chi-square test or Fisher exact test, where appropriate. Genotypes which were statistically significant by univariate analysis were further analysed by logistic regression to reveal the most important predictor genotype. Genotype frequencies were tested by Hardy-Weinberg equilibrium law. A statistical significance was considered when the P < 0.05.
| ~ Results|| |
A total of 290 patients were included in this study, 117 males and 93 females. H. pylori infection was confirmed in 210 patients; 90 patients were diagnosed with chronic gastritis (Group 1), 75 patients were diagnosed with PUD (Group 2) and 45 patients were diagnosed with GC (Group 3). In addition to 80 H. pylori-negative subjects with no significant endoscopic or pathological findings were selected as controls. Other patients' characteristics are summarised in [Table 2]. No significant statistical differences were observed between H. pylori-positive patients and control group regarding age, sex, smoking or alcohol consumption.
|Table 2: The demographic and clinical characteristics of the study population|
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Association between toll-like receptors single nucleotide polymorphisms and Helicobacter pylori infection
The genotypes and alleles frequencies of the studied six SNPs are shown in [Table 3] and [Table 4]. The distributions of each of the six SNPs were tested by goodness-of-fit test for their deviation from expected values. According to Hardy-Weinberg equilibrium law; all SNPs fitted the HWE except for TLR2-rs3804099 and that was excluded from later analysis. The distribution of TLR2-rs3804100 showed higher frequencies of the TT genotype than the TC/CC genotypes. However, no significant statistical difference was observed between H. pylori infected patients and the controls [Table 3]. Furthermore, the differences were statistically insignificant when comparing each patient group with the controls [Table 4]. Both TLR4-rs4986790 and-rs4986791 SNPs showed significant difference (odds ratio [OR] = 4.28, 95% confidence interval [CI] = 1.27–14.44, P = 0.009 and OR = 3.47, 95% CI = 1.02–11.83, P = 0.023, respectively) in genotypes distribution between H. pylori-infected patients and the controls [Table 3]. However, this significant difference is restricted to patients with chronic gastritis (OR = 4.33, 95% CI = 1.19–15.81, P = 0.013 and OR = 6.2, 95% CI = 1.64–12.13, P = 0.004, respectively) and GC (OR = 3.57, 95% CI = 0.96–13.31, P = 0.029 and OR = 3.95, 95% CI = 0.94–16.64, P = 0.031, respectively). While, the difference was non-significant between patients with PUD and controls [Table 4]. The distribution of TLR9-rs352140 genotypes showed no significant association with the overall H. pylori infection status (P > 0.5). However, TLR9-rs352140 TT genotype was found significantly higher among chronic gastritis patients compared to the control group (OR = 2.87, 95% CI = 1.07–7.69, P = 0.018). The distribution of TLR10-rs10004195 SNP showed significant association with the H. pylori infection status (OR = 0.50, 95% CI = 0.25–0.99, P = 0.023), especially in patients with chronic gastritis and PUD (OR = 0.26, 95% CI = 0.10–0.71, P = 0.004 and OR = 0.26, 95% CI = 0.09–0.76, P = 0.007 respectively) [Table 3] and [Table 4]. TLR4 and TLR10 SNPs which were statistically significant by univariate analysis were further analysed by logistic regression to reveal the most important predictor genotype. Logistic regression revealed that the only significant SNP predictor for the disease was TLR10-rs10004195 (P < 0.001, wald 21.6).
|Table 3: The association between toll-like receptors genotypes and alleles with Helicobacter pylori infection status|
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|Table 4: Association between toll-like receptors genotypes and alleles with the clinical outcome of Helicobacter pylori infection|
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| ~ Discussion|| |
The outcome of H. pylori infection is supposed to be regulated by three main factors; bacterial virulence constituents, host immunity and genetic susceptibility in addition to gastric environmental factors. The host's innate immunity is the first line of defence against the invading pathogens. In the case of H. pylori, gastric epithelial cells are considered the first line of innate immunity against infection. The Gastric epithelial cells were found to express pattern recognition receptors (PRRs) as TLRs. These TLRs can recognise different bacterial components and trigger many cell signalling cascades playing an essential role in the recruitment of adaptive immunity and host's inflammatory response. Recent studies have investigated TLRs genetic polymorphisms and the possible link with H. pylor i-associated gasteropathies. TLR2 is one of the best characterised TLRs. It can recognise bacterial LPS, lipoproteins and peptidoglycan resulting in the production of several pro-inflammatory cytokines and initiation of Th1 immune response., Several studies reported an increase of TLR2 expression in gastric mucosa as a result of H. pylori infection as well as in H. pylori-associated GC., In the present study, two common TLR2 SNPs were investigated; TLR2-rs3804099 and TLR2-rs3804100. The results failed to identify any significant association between either of these two SNPs and H. pylori infection or H. pylori-associated gastric diseases. These results are consistent with two previous studies conducted in Japanese and Thai patients., Contrariwise, many previous studies reported significant association between these SNPs and H. pylori infection and its sequels, especially GC., In humans, TLR4 was the first identified receptor out of all TLRs. Alongside TLR2, TLR4 can recognise bacterial LPS and activate both innate and adaptive immunity through NF-κβ pathway in gastric epithelial cells. TLR4 expression was found to be up-regulated at the apical and basolateral membrane of gastric epithelial cells as a result of H. pylori infection. Therefore, gene polymorphisms that resulted in hypo-reactive or reduced functioning TLR4 may be associated with a reduced immune response, eventually lead to a persistent infection. In this studyTLR4-rs4986790 and TLR4-rs4986791 were investigated. Both SNPs had a significant association with H. pylori infection [Table 3]. However, this association was insignificant among the PUD group compared to the controls [Table 4]. These results were concordant with the earlier studies in different ethnic populations in whom these TLR4 gene polymorphisms were associated with susceptibility to H. pylori infection.,,,, On the other hand, other studies revealed no significant association between TLR4 polymorphisms and H. pylori infection or its long term consequences., A probable explanation for this association was reported by Uno et al., where, TLR4-rs4986790 GG genotype gene variant may functionally diminish the TLR4 binding affinity to H. pylori LPS resulting in a diminished the adaptive immune response offered by wild type TLR4 with subsequent development of chronic inflammation and carcinogenesis. Furthermore, Moura et al., have reported an interesting observation where these TLR4 gene variants were significantly associated with infection by the more virulent H. pylori CagA-positive strains. Therefore, the wild-type TLR4 may be considered as an inhibitor of these virulent H. pylori strains. TLR9 is an endosome-trans-membrane receptor that able to recognise microbial DNA. On H. pylori DNA recognition by TLR9, either pro-inflammatory or anti-inflammatory responses may be induced. This dichotomous role of TLR9 in the case of H. pylori infection is highly influenced by the gastric microenvironment. The anti-inflammatory responses were recorded mainly during the acute phase that enable H. pylori to establish persistent infection., The results of the current study showed a significant association between TLR9-rs352140 SNP and H. pylori-associated chronic gastritis, which mean that the occurrence of this SNP may potentiate the anti-inflammatory response and increase the incidence of chronic gastritis. This conclusion is consistent with many earlier studies., On the contrary, Zhao et al. reported no significant association between TLR9 SNP and H. pylori-related diseases. However, further studies are still required to identify precisely micro-environmental conditions that trigger either the pro-inflammatory or the anti-inflammatory response by TLR9. TLR10 is expressed on the surface gastric epithelial cells forming heterodimers with TLR2 and/or TLR6. These heterodimers recognise multiple distinct patterns of H. pylori LPS and diversify innate immune recognition., Few studies have investigated TLR10 SNP association with H. pylori-related gastric pathology. This study indicated that TLR10-rs10004195 was identified to be significantly associated with the susceptibility to H. pylori infection where the rate of variant TT genotype and T allele were found to be significantly lower among both of chronic H. pylori infection and PUD groups compared to the controls. These results are consistent with a previous study conducted in the European population. Furthermore, Tang et al., reported that rs10004195 T allele was significantly associated with a decreased risk of H. pylori infection and atrophic gastritis among the Chinese population. This difference in the inflammatory response and outcomes may be mediated by a modulation of the signalling pathway that leads to the downstream expression of the inflammatory cytokines as IL-1 β, IL-1α, IL-6, IL-8, IL-10, and tumor necrosis factor alpha. On the other hand, TLR10-rs10004195 T allele showed no significant protective effect against H. pylori infection or its long-term consequences in Malaysian patients. That statistical analysis of the distribution of the studied TLR SNPs among the different H. pylori infection outcomes revealed that TLR4 SNPs showed no significant differences in the distribution among PUD and GC patients compared to chronic gastritis group. While TLR9 TT genotype was found to be significantly less prevalent among PUD and GC patients compared to the chronic gastritis group (P = 0.007 and 0.018, respectively). Furthermore, TLR10 TT genotype was found to be significantly more prevalent among GC patients compared to the chronic gastritis group (P = 0.003).
The results of the present study revealed more association between TLR SNPs and chronic gastritis, whether increasing its risk (in case of TLR4 and TLR9) or provide a protective effect (in case of TLR10) while PUD had the lowest association with these SNPs. It seems that TLR SNPs may affect H. pylori infection susceptibility and its outcome. However, many other factors should be considered, especially the ethnic factors that may modify this effect. Furthermore, these results should be cautiously interpreted, and further studies performed on a larger scale are needed for confirmation of the relationship of these TLRs SNPs with the H. pylori infection susceptibility and its long-term consequences. However, the awareness about the predominant hosts' TLRs genotypes in a certain region may enable better prediction of H. pylori infection outcomes among those population.
Financial support and sponsorship
This study was funded by the Deanship of Scientific Research, Taif University, KSA (project number 1-439-6081).
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]