Association of H pylori cagA and vacA genotypes and IL-8 gene polymorphisms with clinical outcome of infection in Iranian patients with gastrointestinal diseases

Abstract

AIM: To find out if a functional promoter polymorphism in the IL-8 gene along with cagA status and polymorphisms in vacA gene influence the type of diseases in Iranian patients infected by H pylori.

METHODS: IL-8 -251 A/T polymorphism was genotyped by oligonucleotide allele specific PCR (ASO-PCR) in a sample of 233 patients with H pylori infection undergoing upper gastrointestinal endoscopy. The presence of cagA gene and polymorphisms in vacA gene was also determined by PCR. Association of these genetic polymorphisms with the development of gastritis, peptic ulcers as well as gastric cancer was tested.

RESULTS: When the patients with different clinical manifestations were compared according to the presence of cagA gene or various vacA genotypes, only the vacA genotypes were significantly different among gastritis, peptic ulcer and gastric cancer patients (χ² = 17.8; P = 0.001). Furthermore, there was a significant difference in the frequency of IL-8 -251 A/T genotypes between patients with gastric cancer and benign diseases (χ² = 10.47; P = 0.005).

CONCLUSION: The IL-8 -251 A/T polymorphism and the polymorphisms in H pylori vacA gene are involved in limiting the infection outcome to gastritis and peptic ulcer or in favoring cancer onset in Iranian patients.

Key Words: Interleukin 8; H pylori; Gastric cancer; Peptic ulcer; Polymorphism

INTRODUCTION

Infection with H pylori has plausible associations with a variety of clinical outcomes, including chronic gastritis, peptic ulcer and gastric cancer[1-4]. Variation in the clinical outcome of H pylori induced pathology is multifactorial, involving a complex interplay between the host immune responses, pathogen virulence factors, and niche characteristics. Many putative virulence factors have been identified in H pylori that contribute to its pathogenesis. The 128-kDa cytotoxin-associated gene encoded antigen A (cagA)[5] and vacuolating cytotoxin antigen gene (vacA)[6] are known as the most important ones. cagA gene was identified as a strain-specific H pylori gene and has been recognized as a marker for strains that confer increased risk for peptic ulcer disease[7-8] and gastric cancer[9]. The cagA gene is present downstream of a 40-kb cluster of virulence genes known as the cag pathogenicity island (cag-PAI). These genes encode a type IV secretion system that forms a syringe-like structure to translocate the immunodominant cagA protein into the gastric epithelial cells. cag-PAI has also been implicated in the induction of IL-8 in cultured gastric cells[10]. This property contributes to the proinflammatory power of the strains and thus to their virulence capability. The difference between H pylori strains in virulence capability is also dependent on the expression of vacA (87 kDa), which is toxic to epithelial cells in vitro[11-13]. Moreover, mice which were administered vacA orally developed gastric ulcers[14]. Unlike the cagA, vacA gene is conserved among all H pylori strains, although significant polymorphism exists in its gene[15]. vacA alleles possess one of two types of signal regions, s1 or s2, and one of two mid-regions, m1 or m2, occurring in all possible combinations. The vacA signal region encodes the signal peptide and the N-terminus of the processed vacA toxin: type s1 vacA is fully active, but type 2 has a short N-terminus extension that blocks vacuole formation[16]. vacA s2 strains are rarely isolated from patients with peptic ulcers or gastric adenocarcinoma[15]. The vacA mid-region encodes part of the toxin cell binding domain. Vacuolating activity is higher in s1/m1 genotypes than in s1/m2 genotypes, and is absent in s2/m2 genotypes[16]. Consequently, vacA s1/m1 genotypes are more frequently associated with peptic ulceration and gastric carcinoma[17,18]. The genetic heterogeneity in immune responses among individuals is another important factor which determines the clinical outcome of H pylori infection. Support for this consideration is provided by the low frequency of gastric cancer in some developing countries in spite of the paradoxically high prevalence of H pylori infection in those countries[19,20]. Up to now, there are several reports indicating the association of IL-1β, tumor necrosis factor α (TNF-α), and IL-10 gene polymorphisms with an increased risk of developing gastric atrophy, hypochlorhydria, and non-cardia gastric cancer[21-23]. Due to the roles which are played by IL-8 in the pathogenesis of H pylori infection, the IL-8 gene is one of the most important candidate host genes in determination of the outcome of H pylori infection. This cytokine is produced by gastric epithelial cells as an early response to H pylori virulence factors, such as cagA[10]. IL-8 is also a major host mediator involved in neutrophil and phagocyte chemotaxis and activation[24,25], thereby causing mucosal damage by releasing reactive oxygen radicals[25]. It is therefore tempting to speculate that mucosal IL-8 production due to H pylori infection may be an important factor in the immunopathogenesis of peptic ulcer diseases and may also be relevant in gastric carcinogenesis[26]. Interestingly, previous studies have suggested that the production of IL-8 is genetically determined and neutrophils from individuals who are homozygous for the AA genotype at the -251 position demonstrated a trend toward higher levels of IL-8 production in response to lipopolysaccharides than those without the allele[27]. More recently, Ohyauchi et al reported that IL-8 -251 A/T polymorphism influences the susceptibility of

H pylori related gastric diseases in the Japanese population[28]. Furthermore, in an H pylori infected Chinese population the risk of gastric cancer was also significantly elevated in patients with the IL-8-251 AA genotype[29]. Considering the above information, the aim of this study was to look for an association between IL-8 -251 A/T polymorphism, vacA genotypes, the presence of cagA gene and clinical outcome of H pylori infection in Iranian patients.

MATERIALS AND METHODS

Patients and Bacterial strains

In the present study 298 patients were classified at the time of endoscopy into those having gastritis (n = 199), gastric ulcer (n = 12), duodenal ulcer or duodenitis (n = 67) and non-cardia gastric carcinoma (n = 20). This classification was also confirmed by histological examinations. These patients have been referred for upper gastrointestinal endoscopy to the Gastroenterology Section of the University Hospitals (Namazi and Shahid Faghihi) of Shiraz University of Medical Sciences between 2002 and 2005. Patients who had received non-steroidal anti-inflammatory drugs were excluded. H pylori strains were successfully isolated from the gastric biopsies of 286 patients (150 males, 136 females; median age 45.3 ± 16.6 years). The present study was approved by the local ethics committee.

Bacterial culture and histological examination

Biopsy specimens were taken from the antrum and corpus of the stomach. These specimens were used for the rapid urease test, bacterial culture, and histological assessment. After 5 d of culture on selective agar plates, the organisms were identified as H pylori by Gram staining, colony morphology, and positive oxidase, catalase and urease reactions.

Preparation of patients and H pylori genomic DNA

After 3-5 d of culture, H pylori colonies were pooled from the plates and washed using phosphate-buffered saline. H pylori genomic DNA was prepared after bacterial cell lysis using SDS and proteinase K solution and phenol-chloroform extraction. Patients genomic DNA was extracted from EDTA anticoagulated peripheral blood leucocytes using a salting out method. The DNA samples were maintained at -70°C until use in polymerase chain reaction (PCR).

Analysis of IL-8 and bacterial vacA and cagA genotypes

All primer sets used were selected from the published literature and are shown in Table 1. An allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) was used to detect the polymorphism at position -251 of the IL-8 gene[30]. As an internal control, the β-globin specific primers were included in the ASO-PCR (Table  1). For IL-8 genotyping, 10 μL of PCR reaction mixture consisting of 250 ng of genomic DNA, 200 μmol/L dNTPs, 2.25 mmol/L MgCl₂, 1 × Taq DNA polymerase buffer, 2 units of Taq DNA polymerase (Boehringer Manheim, Germany), 10 pmol of each test primer and 5 pmol of internal control primers were employed. Then, a touch-down procedure was followed that consisted of 25 s at 95°C, annealing for 45 s at temperatures decreasing from 68°C (four cycles) to 61°C (20 cycles), and an extension step at 72°C for 40 s. The annealing temperature for the remaining 5 cycles was 58°C for 40 s. For cagA and vacA genes the PCR was performed using a thermal cycler (Master Cycler; Eppenderof, Germany) under the following conditions: an initial denaturation for 5 min at 94°C, 35 cycles of 60 s at 94°C, 60 s at 57°C and 60 s at 72°C, with a final extension step at 72°C for 5 min. The PCR system for cagA contained 10 × PCR buffer, 2.5 μL; MgCl₂, 1.2 mmol/L; dNTP, 200 μmol/L; cagA specific primers, 10 pmol; Taq DNA polymerase, 1.0 U; and the DNA template, 50 ng. To confirm the identification of the bacteria as H pylori, 10 pmol of glmM specific primers were included in each PCR reaction for cagA gene (glmM is a conserved gene formerly known as ureC). For detection of vacA polymorphisms a multiplex PCR was performed using the specific primers for amplification of s and m genes (Table 1). The PCR system for vacA genotyping was similar to cagA genotyping. PCR products were examined by 2% agarose gel electrophoresis and photographed using an ultraviolet reflection analyzer.

Table 1 Primer sequences used for detection of cagA gene status and IL-8 or vacA gene polymorphisms.

Locus	Primers	Size (bp)
IL-8 -251	Common primer, 5’-tgc ccc ttc act ctg tta ac-3’	336
	A allele, 5’-cca caa ttt ggt gaa tta tca at-3’
	T allele, 5’-cca caa ttt ggt gaa tta tca aa-3’
β-globin	5’- aca caa ctg tgt tca cta gc -3’	100
	5’- caa ctt cat cca cgt tca cc -3’
cagA	5’-aat aca cca acg cct cca ag-3’	400
	5’-ttg ttg ccg ctt ttg ctc tc-3’
vacA
S1 and s2	5’-ctg ctt gaa tgc gcc aaa c-3’	s1 = 259
	5’-atg gaa ata caa caa aca cac-3’	s2 = 286
m1 and m2	5’-gcg tct aaa taa ttc caa gg-3’	m1 = 570
	5’-caa tct gtc caa tca agc gag-3’	m2 = 645
glmM	5’-aag ctt tta ggg gtg tta ggg gtt t-3’	294
	5’-aag ctt act ttc taa cac taa cgc-3’

Statistical analysis

Fisher’s exact test and the χ² test were used to analyze the data from different disease groups. All tests were performed two tailed with a confidence interval (CI) of 95%. A P-value of less than 0.05 was accepted as statistically significant. The Statistical Package for the Social Sciences (SPSS) version 11.5 was used for statistical analysis.

RESULTS

Prevalence of the cagA and vacA gene in different disease groups

Table 2 shows the distribution of the 286 strains according to their cagA and vacA types in different patient groups. To detect the cagA subtype of H pylori, cagA specific PCR was performed on extracted bacterial DNA from patients with gastric diseases. Amplified cagA DNA fragments were detected in 219/286 (76.6%) of our H pylori. Sixty seven patients (23.4%) were cagA-, although they were glmM+ or vacA+. When the patients with different clinical manifestations were compared according to the presence of the cagA gene (Table 2), insignificant differences in cagA status were found among patients with peptic ulcer, gastritis and gastric cancer (χ² = 1.9; P = 0.38). Also, when the frequency of cagA+ and cagA- subtypes were compared among patients with gastritis and gastric ulcer (χ² = 1; P = 0.3), gastritis and peptic ulcer (χ² = 0.75; P = 0.38), gastritis and cancer (χ² = 0.61; P = 0.42) or between peptic ulcer and gastric cancer (χ² = 0.01; P = 0.75), the difference always remained insignificant. In the case of vacA gene, three of the four possible combinations of signal sequence and middle-region types were identified. The s1/m1 type was found in 81/264 (30.7%) of the isolates, the s1/m2 type in 110/264 (41.7%) of the isolates, and the s2/m2 type in 73/264 (27.7%) of the isolates. The distribution of vacA genotypes (s1/m1, s1/m2 or s2/m2) were significantly different among peptic ulcer, gastritis and gastric cancer patients (χ² = 17.8; P = 0.001). Interestingly, the difference in distribution of three different vacA genotypes between patients with gastric cancer and gastritis (χ² = 12.57; P = 0.0018) was more significant than the difference in distribution of vacA genotypes between patients with gastric cancer and peptic ulcer (χ² = 7.97; P = 0.018). In fact, the frequency of s1m1 genotype was notably higher in gastric cancer patients (66.7%) compared to those with gastritis or peptic ulcer (26.5% and 32.3%, respectively). Furthermore, similar to previous reports[15], there is a strong statistical linkage between the s1 genotype of vacA and the presence of the cag island (χ² = 27.95; P = 0.0000001; OR = 4.99, 95% CI = 2.61-9.58). Similarly, the s2 genotype is associated with the lack of the cag island. In fact, strains that are cag+ are more likely to possess the vacA s1 allele than cag- strains.

Table 2 IL-8 -251 A/T polymorphism, cagA status and vacA gene polymorphism in patients with gastric diseases.

Locus	Genotype	Patients Gastritisn(%)	Peptic Ulcern(%)	Gastric Cancern(%)	Pvalue
IL-8 -251	AA	22 (14.4)	14 (23.0)	9.0 (47.4)	0.013
	AT	74 (48.4)	28 (45.9)	6.0 (31.6)
	TT	57 (37.3)	19 (31.1)	4.0 (21.1)
cagA	+	148 (74.4)	54 (80.6)	17 (85.0)	0.39
	-	51 (25.6)	13 (19.4)	3.0 (15.0)
vacA	s1m1	48 (26.5)	21 (32.3)	12.0 (66.7)	0.001
	s1m2	74 (40.9)	33 (50.8)	3.0 (16.7)
	s2m2	59 (32.6)	11 (16.9)	3.0 (16.7)

Prevalence of the IL-8 -251 A/T polymorphism in different disease groups

The allelic frequencies of IL-8 -251 A/T polymorphism and genotype distributions are given in table 2. The IL-8 -251 A/T polymorphism showed no evidence of deviation from the Hardy-Weinberg equilibrium, with a non-significant χ² value (χ² = 0.05, P = 0.4). Interestingly, there was a significant difference in the frequency of IL-8 -251 A/T genotypes between patients with gastric cancer and those with benign diseases (χ² = 10.47; P = 0.005). Moreover, when the patients were categorized to high producer (AA) and intermediate + low producer (AT + TT) genotypes, a more meaningful difference in the frequency of IL-8 -251 A/T genotypes between patients with gastric cancer and benign diseases was noticeable (OR = 4.45, 95% CI = 1.53-12.94; χ² = 8.58, P = 0.003). In fact, the prevalence of AA genotype in gastric cancer patients was 47.4% compared to 16.8% in benign diseases. While comparing different patient groups, no significant differences were demonstrated in frequencies of IL-8 -251 A/T genotypes between patients with peptic ulcer and gastritis (χ² = 2.4; P = 0.25) or gastric cancer (χ² = 4.22; P = 0.12). However, IL-8 -251 A/T polymorphism showed a significant difference between patients with gastric cancer and gastritis (χ² = 12.5; P = 0.001).

DISCUSSION

After exposure to H pylori, the clinical manifestations are variable and depend on host and pathogen factors. There is no information on the prevalence and role of H pylori genotypes and/or the role of IL-8 -251 A/T genotypes in the disease outcome of Iranian patients. Therefore, in the present study, we determined the presence of the cagA gene (as a marker of cag pathogenicity island) and the genotypes of vacA gene in the infecting strains along with the distribution of host IL-8 genotypes in relation to the occurrence of different clinical manifestations in Iranian patients with H pylori infection. It has been shown that exposure of gastric epithelial cells to cag+ H pylori strains can activate the proto-oncogenes c-fos and c-jun, a crucial step in the development of H pylori-related neoplasia[31]. The presence of cagA has been statistically associated with duodenal ulceration, gastric mucosal atrophy, and gastric cancer[7-9], although some studies deny this association[32-34]. Audibert and her colleagues reported that cagA status is not sufficient to predict the IL-8 induction ability of H pylori and is not correlated with the presence or absence of ulcer[35]. In a series of patients from Taiwan, the presence of cagA gene in the PAI also showed no relationship to the type of disease and/or the histological features of the patients[36]. The results of the present study also shows that the prevalence of cagA is not significantly different among different disease groups (P > 0.05), which is in accordance with the results of other Asian countries. Therefore, the cag-PAI may not be the principal virulence factor, as suggested by the absence or sporadic distribution of the cag-PAI genes among strains from varied clinical outcomes. However, considering the high prevalence of cag+ strains in Iranian patients (76.6%), the relationship of cag status with disease type is more difficult to establish in our population. Therefore, larger sample sizes are recommended for such studies.

Furthermore, the lack of association may be due to the fact that the development of cancer or ulcer disease is a complex process that also involves factors other than the cag-PAI, such as vacA. Certain vacA genotypes causing a high vacuolating activity are correlated with increased disease severity in humans[15]. Several studies have also shown that the presence of vacA is associated with peptic ulcer diseases[14,17-18]. The vacA gene displays a considerable polymorphism, especially in the signal region (genotypes s1 and s2) and in a mid region (genotypes m1 and m2). Vacuolating activity is higher in s1/m1 genotypes than in s1/m2 genotypes, and is absent in s2/m2 genotypes[15]. Consequently, vacA s1/m1 strains cause more direct epithelial damage and are more frequently associated with peptic ulceration and gastric carcinoma[9,17,18]. The results of the present study also showy a significant difference in vacA genotype distribution between gastric cancer and gastritis (P = 0.0018) or peptic ulcer (P = 0.018) patients. The marked increase of s1m1 genotype in gastric cancer patients (66.7%) compared to those in patients with gastritis (26.5%) or peptic ulcer (32.3%) confirms the pathogenic role of this virulence determinant in Iranian patients. However, different disease outcomes were encountered in subjects infected with H pylori strains sharing the same virulent vacA genotype, s1m1. The different outcomes of H pylori infection may depend not only on other bacterial factors but also on the different genetic background of the host. Concerning host genetic factors, Thye et al performed a genome wide screen analysis to identify the genetic factor(s), which define susceptibility to H pylori infection[37] and suggested the presence of a possible linkage with chromosomes[4]. Considering the location of the human IL-8 gene on chromosome 4 (4q13-q21), the results of their study may support the hypothesis that the IL-8 gene polymorphism is probably associated with H pylori induced gastrointestinal diseases. Interestingly, the inheritance of the IL-8 -251A allele was associated with progression of gastric atrophy in patients with H pylori infection and increased the risk of gastric cancer in Japanese and Chinese people[28,29,38]. Our results also indicate that gastric cancer is significantly associated with the functional polymorphism in the promoter region of the IL-8 gene. Specifically, individuals genetically predisposed to produce more IL-8 are at a higher risk of developing gastric cancer. The finding that there was an increased risk of gastric cancer in high IL-8 producers was in agreement with the concept that IL-8 may influence the risk of developing gastric cancer by altering the quality and vigor of inflammatory responses produced by the host after exposure to H pylori. In addition, IL-8 stimulated neutrophils to synthesize active radicals such as nitric oxide[25]. These radicals by their mutagenic potential[39] could cause mutations in gastric epithelial cells. In addition, IL-8 by inducing angiogenesis would be one of the important factors in gastric carcinogenesis. In support of this hypothesis, the expression of IL-8 has been associated with increased vascularization and poor prognosis in gastric carcinoma[40,41]. Thus, inheritance of the high producer allele of IL-8 (carriers of -251 A allele) may induce chronic gastritis, which may then be followed by the development of gastric cancer.

In conclusion, similar to studies performed in China and Japan, the association between cagA positivity and virulence of H pylori strains was equally frequent among Iranian patients with different disease types. Moreover, the present study provides further evidence that in addition to genetic polymorphism of the vacA gene in the pathogen, genetically determined differences in IL-8 production via promoter polymorphisms could contribute to individual susceptibility to gastric cancer development after H pylori infection in Iranian patients.

ACKNOWLEDGMENTS

We wish to thank Professor Mahmud Vesal for critical reading of the manuscript. Thanks are also due to Mr. Mohsen Hoseini-Farzad, Mr. Behrouz Gharesi-Fard, Mr. Micheal Shamoon and Mr. Hadi Farsiani of the departments of Immunology and Microbiology for their technical assistance.