Helicobacter pylori cag Pathogenicity Island cagL and orf 17 Genotypes Predict Risk of Peptic Ulcerations but not Gastric Cancer in Iran

Gastric cancer (GC) is the third common cancer that ends up death in the world* with different incidence rate based on geographical areas (Parkin, 2004). The high incidence of GC has geographical distribution in Asia especially in Iran (Kelley and Duggan, 2003; Sadjadi et al., 2003). The GC has high rate in male and is the third cancer after breast and colorectal cancer in female in Iran (Malekzadeh et al., 2009; Mousavi et al., 2009). Ardabil province is located in Northwestern Iran, is reported as area with the high incidence rate of gastric adenocarcinoma (Sadjadi et al., 2003). The concrete reasons for prevalence of GC, which varies in different geographical areas, remain unknown. However, several studies have been shown that interaction between host and environmental factors, especially Helicobacter pylori (H. pylori) infection may play a remarkable role (Covacci et al., 1993; Atherton et al., 1995; Peek and Blaser, 2002; Yamaoka, 2010). This bacterium is found in more than 50 % of the world


Introduction
Gastric cancer (GC) is the third common cancer that ends up death in the world* with different incidence rate based on geographical areas (Parkin, 2004).The high incidence of GC has geographical distribution in Asia especially in Iran (Kelley and Duggan, 2003;Sadjadi et al., 2003).The GC has high rate in male and is the third cancer after breast and colorectal cancer in female in Iran (Malekzadeh et al., 2009;Mousavi et al., 2009).Ardabil province is located in Northwestern Iran, is reported as area with the high incidence rate of gastric adenocarcinoma (Sadjadi et al., 2003).The concrete reasons for prevalence of GC, which varies in different geographical areas, remain unknown.
However, several studies have been shown that interaction between host and environmental factors, especially Helicobacter pylori (H.pylori) infection may play a remarkable role (Covacci et al., 1993;Atherton et al., 1995;Peek and Blaser, 2002;Yamaoka, 2010).This bacterium is found in more than 50 % of the world Department of Biology, Faculty of Sciences, University of Mohaghegh Ardabili, Ardabil, Iran *For correspondence: slatifin@ yahoo.com

Genotypes Predict Risk of Peptic Ulcerations but not Gastric Cancer in Iran
Negin Raei, Saeid Latifi-Navid*, Saber Zahri population and it is a well-known human pathogen.H. pylori is the major cause of acute or chronic gastritis, peptic ulcerations (PU) disease, gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma.In most of cases the infection remains asymptomatic during life, just 20% develops peptic ulcer or gastric carcinoma (Blaser, 1992a;Blaser, 1992b;1994;Blaser and Parsonnet, 1994;Parsonnet et al., 1997;McColl and El-Omar, 2002;Sepulveda and Graham, 2002;Kapadia, 2003;Matsuhisa et al., 2003).The cag pathogenicity island (cag PAI) of H. pylori is an important virulence factor that involves several genes with different function in the H. pylori (Crabtree et al., 1995a;Crabtree et al., 1995b;Sharma et al., 1995;Tummuru et al., 1995;Censini et al., 1996;Figura and Valassina, 1999;Asahi et al., 2000;Stein et al., 2000;Megraud, 2001).Adherent of bacterium to gastric epithelial cells is critical to initiate the gastric inflammatory response with different clinical outcomes in humans (Hessey et al., 1990;Logan, 1996;Segal et al., 1996;Rieder et al., 1997).CagL (a VirB5 ortholog) is a bacterial factor that is known as a pilus protein (Backert et al., 2008;Backert and Selbach, 2008;Backert et al., 2011), Integrin α5β1 is a receptor on gastric epithelial cell that CagL binds to; by its arginineglycine-aspartate (RGD) motif, this interaction activates α5β1 receptor and facilitates the delivery of CagA oncoprotein through type 4 secretion system (T4SS) into the epithelial cell (Parsonnet et al., 1997;Kwok et al., 2007;Backert and Selbach, 2008;Backert et al., 2011;Shaffer et al., 2011).CagL is associated with two other cag PAI proteins; CagI and CagH.All the three factors are essential for the injection of CagA into the epithelial cell and T4SS pili formation, studies have also suggested that pilus dimension might be regulated by CagH.In addition, cagH is important for induction of IL-8 secretion by epithelial cells which mediates increased risk of developing GC (Kwok et al., 2007;Shaffer et al., 2011).cagG is another factor that is located upstream of the cagA gene.The cagG has also been shown some homology to the adherence-related or motility-related genes of other bacteria in bioinformatics study (Censini et al., 1996;Weel et al., 1996;Perez-Perez et al., 1999;Audibert et al., 2001;Li et al., 2001).The orf17 of H. pylori shows some homology to a gene from Dickeya zeae that 36% identity is reported in the BLASTp studies.Dickeya zeae, is the aerobic/anaerobic phytopathogen bacterium that causes soft rot disease in a broad range plants species, especially many crops; this bacterium causes bacterial foot rot and it is reported in many Asian countries particularly China which the disease mostly begins at the ligulus (Samson et al., 2005;Zhou et al., 2011).The purpose of this study was to find out the distribution of four target genes of cag pathogenicity island (cagH, cagL, cagG, and orf17) in H. pylori genome and their relationship with gastrointestinal disease in Iran.

Collection of tissue specimens
The tissue specimens were collected over seven years from 2007 to 2014 in Iran; from patients with various gastrointestinal diseases as mentioned in Table 1.

H. pylori isolation and culture
Antrum and body biopsies were used for H. pylori culture.Biopsies were cultured on selective Brucella agar (Merck, Germany) involving 5% sheep blood, 10 mg/L of vancomycin (Zakaria, Iran), 5 mg/L trimethoprim (MP Biomedicals, France), 2.5 IU/L polymyxin B (MP Biomedicals, France), and 8 mg/L amphotericin B (Bristol-Myers Squibb, USA).Cultures were incubated at 37°C under microaerophilic condition for 3-7 days.Bacterial isolates were identified as H. pylori on the basis of Gram-stained morphology and positive urease, catalase, and oxidase tests.Bacterial isolates were harvested in brain heart infusion broth (Merck) enriched with 20% glycerol and 10% inactivated horse serum and stored at -70°C.

Histological assessment and classification
All biopsies were taken from the gastric body (corpus) and antrum of patients with different gastrointestinal disease, and were used for histopathological examination.The biopsy specimens were initially formalin-fixed and paraffin-embedded.Sections of 4μm were obtained and stained with hematoxylin-eosin, Giemsa, and Alcian blue-periodic acid Shiff (pH 2.5).By the use of Sydney system, GC was classified and graded (Table 1) (Dixon et al., 1996).

DNA extraction
Genomic DNA was extracted from the H. pylori culture positive gastric biopsy specimens, using DNGTM plus kit (CinnaGen, Tehran, Iran) according to the manufacturer's instructions.Extracted DNA was stored at -20˚C until used for polymerase chain reaction (PCR) amplification.

PCR amplification
The final identification of H. pylori was confirmed by using 16S rDNA gene PCR by species-specific primers as shown in Table 2 (Lu et al., 2002).The second PCR was done in order to detect the target genes which were cagH, cagL, cagG, and orf17 (Table 2).The PCR reaction was carried out in a 30-μl reaction mixture involving MgCl2=1 µl, PCR buffer=3 µl, dNTP mix=0.5 µl, primers (reverse and forward mixture)=1 µl, enzyme=0.2µl, template DNA=5 µl, and distilled water=19.3µl (total volume=30 µl).The genes amplification conditions were as follows: predenaturation was carried out at 95°C for 45 s, followed by 30 cycles of 94°C for 45s, 72°C for 45s, and a final extension at 72°C for 7 min.The PCR products were developed under ultraviolet (UV) light after electrophoresis on 1% (w/v) agarose gel.H. pylori IRANL15 strain and a no-template reaction used as positive and negative controls in each PCR experiment, respectively.
Amplified fragments of each gene from six isolates were purified and sequenced with both forward and reverse primers by using BigDye technology on an ABI3700XL DNA sequencer (Applied Biosystems).The BLAST program (http://www.ncbi.nlm.nih.gov) was used to match the nucleotide sequences with the published sequences in GenBank.

Statistical analysis
For statistical analysis, the SPSS 18.0 software was used (SPSS Inc, Chicago, Illinois, USA).Chi square and Fischer's exact tests were used to analyze the of H. pylori genotypes with histopathological and clinical outcomes.A P-value of<0.05 indicated significance.A simple and multiple logistic regression were used to calculate the odds ratio (OR) and 95% confidence interval (CI) of the clinical variables.The OR and CI were used to estimate the risk of PU and GC in the current study.

Discussion
The presence of H. pylori in the gastric mucosa has been known as an essential risk factor of different gastrointestinal disease including; NAG, PU, and GC.H. pylori has several virulence factors that might take part in mucosal damage (Mahdavi et al., 2002;Erzin et al., 2006;Kusters et al., 2006;Miernyk et al., 2011;Yeh et al., 2011;Shiota et al., 2012).This study investigated the relevance of various virulence factors (cagH, cagL, cagG, and orf17) and their relationship with sever gastrointestinal disease.The cagH genotype has not been surveyed on genomics level; however, we found that the cagH genotype had no relationship with gastrointestinal disease in Iran (P>0.05).Recently, it has been demonstrated that 86.6% of the H. pylori isolates from Indian patients were cagL positive.Other studies in Malaysia, Singapore, and Taiwan demonstrated that more than 85% of the isolates were cagL positive (Schmidt et al., 2010;Yeh et al., 2011).A study on 61 isolates from patients with digestive disease in Iran, showed that 96.7% were cagL positive.This report was concordant with the results from Taiwan, where the 98.6% of the patients were cagL positive, but no remarkable association was detected between the cagL genotype and clinical outcomes (P>0.05)(Kwok et al., 2007;Yeh et al., 2011;Wang et al., 2013;Yadegar et al., 2014).These results are consistent with our results in GC group but not in PU group, while the cagL genotype is remarkably and independently associated with the risk of PU in Iran (P=0.021), but no association was found with the risk of GC (P>0.05).Previously studies showed that cagG mutants were impaired to induce inflammatory response as well as increase proliferation of epithelial cells, and also determined that lack of cagG gene leads to the loss of CagA translocation/phosphorylation (Censini et al., 1996;Tomb et al., 1997;Fischer et al., 2001).Recently reports showed that the loss of cagG genotype has been associated with reduced adherence to epithelial cells (Mizushima et al., 2002).An in vivo study in China has also demonstrated no significant relationship between cagG and clinical outcomes.Several studies have proposed that the lack of cagG genotype led to the loss of induction of IL-8 (Censini et al., 1996;Hsu et al., 2002).However, other studies have proposed that the complete deletion of cagG gene resulted in no reduction in IL-8 induction (Fischer et al., 2001).According to these studies, it is impossible to discriminate whether or not the lack of inflammation with cagG mutant is associated with reduced colonization or loss of T4SS, or both (Figura and Valassina, 1999).A study on 145 H. pylori isolates in China showed that cagG was detected in 91.7% isolates, and 100% of isolates from patients with PU (duodenal ulcer and gastric ulcer) were cagG positive but not statistically significant (P>0.05).cagG genotype was known as a conserve gene in Chines population and there was no significant differences in the frequencies of cagG gene in isolates from patients with various digestive disease.Hsu et al reported that of the 120 isolates from patients with different gastrointestinal disease in Korea 86.7% (104/120) were cagG positive (Hsu et al., 2002).Mizushamia et al used PCR to investigate the distribution of cagG gene in 236 clinical H. pylori isolates in Japan, 97% of isolates were cagG positive.For both isolates from Korea and Japan, cagG positive isolates demonstrated no significant association with gastrointestinal disease (P>0.05)(Mizushima et al., 2002).In other study, the level of gastric mucosal inflammation was compared in the antrum and the body (corpus) of both the cagG positive and negative group.It was illustrated that the level of inflammation was relatively higher in cagG positive than cagG negative group, both in the antrum and the body (P>0.05),but no relationship was detected between cagG genotype and the intensity of gastritis (Shimoyama et al., 1998;Yamaoka et al., 1998).Consistent with the previous studies, we showed that the cagG genotype had no remarkable association with digestive disease in Iran (P>0.05).
The orf17 genotype has also not been studied on genomics level, we showed that orf17 gene had no association with GC group (P>0.05) but there was a remarkable relationship between the presence of orf17 gene and an increased risk of PU in Iran (P=0.015).
This report is the first one regarding the relevance of the H. pylori cagL and orf17 genotypes to PU in Iran.We have proposed that the orf17 and particularly cagL genotypes of H. pylori cag PAI could be as beneficial factors for the risk prediction of PU, but not GC in Iran.In addition, specific H. pylori virulent biomarkers could be used to predict PU or GC risk and selected for H. pylori eradication in patients with NAG.Based these initial results, our future goals are to evaluate several virulence factors simultaneously, in order to clarify the correlation between the virulence factors and gastrointestinal disease.