Helicobacter pylori vacA d 1 Genotype Predicts Risk of Gastric Adenocarcinoma and Peptic Ulcers in Northwestern Iran

H pylori is a microaerophilic gram negative bacterium that is able to colonize and persist within the gastric mucosa layer (Atherton, 2006). Several studies have shown a close relationship between H pylori infection and gastroduodenal diseases, such as chronic active gastritis, PUD, MALT-lymphoma, and GC (Parsonnet et al., 1991). GC (90% adenocarcinomas) is the fourth most common cancer worldwide and the second leading cause of cancer-related deaths (700,000 deaths annually) (Coleman et al., 1993; Parkin et al., 2005). The 63% of all GC are related to chronic H pylori infection (Parkin, 2006). While H pylori infection plays a primary role, genetic and environmental factors modulate its outcome (Peek et al., 2010). Studies have shown that the elimination of H pylori may reduce the risk of PUD and GC but increase the risk of gastroesophageal reflux (GERD), Barrett’s esophagus, and esophageal adenocarcinoma (Blaser, 1999). Considering the significance of H pylori in certain diseases, it is important to identify which strains of H pylori have the potential to increase the risk of GC and PUD (Blaser, 1999). The vacuolating cytotoxin A


Introduction
H pylori is a microaerophilic gram negative bacterium that is able to colonize and persist within the gastric mucosa layer (Atherton, 2006).Several studies have shown a close relationship between H pylori infection and gastroduodenal diseases, such as chronic active gastritis, PUD, MALT-lymphoma, and GC (Parsonnet et al., 1991).GC (90% adenocarcinomas) is the fourth most common cancer worldwide and the second leading cause of cancer-related deaths (700,000 deaths annually) (Coleman et al., 1993;Parkin et al., 2005).The 63% of all GC are related to chronic H pylori infection (Parkin, 2006).While H pylori infection plays a primary role, genetic and environmental factors modulate its outcome (Peek et al., 2010).Studies have shown that the elimination of H pylori may reduce the risk of PUD and GC but increase the risk of gastro-esophageal reflux (GERD), Barrett's esophagus, and esophageal adenocarcinoma (Blaser, 1999).Considering the significance of H pylori in certain diseases, it is important to identify which strains of H pylori have the potential to increase the risk of GC and PUD (Blaser, 1999).The vacuolating cytotoxin A

Helicobacter pylori vacA d1 Genotype Predicts Risk of Gastric Adenocarcinoma and Peptic Ulcers in Northwestern Iran
Zeinab Basiri¹, Reza Safaralizadeh¹*, Morteza jabbarpour Bonyadi¹, Mohammad Hossein Somi², Majid Mahdavi 1 , Saeid Latifi-Navid 3 (vacA) often present in every H pylori strain (Cover et al., 1994).Variation in levels of cytotoxicity is attributed to vacA allelic variation among H pylori strain, which occurs in the signal (s), middle (m) and intermediate (i) regions, each with two different alleles (Atherton et al., 1995;van Doorn et al., 1998): s1 or s2, m1 or m2,and 1i or i2, respectively (Rhead et al., 2007).Recently, a fourth disease-related region between the i and m regions has been indentified and named the deletion (d) region.The d region is divided into d1 (no deletion) and d2 (with a 69 to 81 bp deletion), with d1 identified a risk factor for GC and PUD in Western strains.Nearly all H pylori isolates from East Asia have been found to have a genotype of vacA d1 (Ogiwara et al., 2009).
Almost two-thirds of cases of GC occur in Asia (Nguyen et al., 2008).Iran with a high incidence of GC ranks fourth in Asia, after China, Japan and Korea respectively (Fujisawa et al., 1999;Wang and Wang, 2003;Derakhshan et al., 2004;Yim et al., 2007;Alizadeh et al., 2009).
The prevalence of H pylori infection is 69% in Iran (Nouraie et al., 2009).Phylogenetic analysis performed in Iran has shown that Iranian strains have probably been influenced by genetic exchange from neighboring countries, so that some researchers have suggested Iranian H pylori strains to be a subset of HpEurope population (Latifi-Navid et al., 2010).Another study in Iran has demonstrated that the prevalence of the vacA d1 genotypes in strains representing European ancestry were significantly higher in areas with high incidence of gastric cancer.(Latifi-Navid et al., 2013).East Azerbaijan Province is located in northwestern Iran, where the incidence of GC is high.GC is the leading cause of cancerrelated deaths in males (ASRs=26.0)and the fourth type of cancer after breast, skin and esophagus cancers in females (ASRs=11.6) in this area (Somi et 2008).
The purpose of this study was to investigate the association between the H pylori vacA d1 genotype and disease outcome in infected patients, and to determine whether the H pylori strains in this study mimicked the isolates described in western countries.

Study location and patients
All patients Over 16 years of age referred to the department Endoscopy at Imam Reza Hospital in Tabriz, Iran were given a questionnaire to collect demographic data (age, gender, nationality and language).Individuals who had received non-steroidal anti-inflammatory drugs or anti-Helicobacter therapy at least 3 months prior to endoscopy were excluded (Willen et al., 2000), as were those meeting the criteria listed in Table 1 (Westbrook et al., 2005).Antral gastric biopsies were collected from 115 patients with different gastroduodenal diseases.Informed consent was obtained directly from each patient under protocols approved by the hospital's ethics committee.The sample consisted of 58 males and 57 females with a mean age of 52.9 years (range, 19 to 90 years).

Endoscopy and biopsy sampling
For each patient, at least two biopsy specimens for the rapid-urease test and histology examination, and an additional biopsy specimen for PCR analysis were taken from the antral mucosa Biopsy specimens for PCR analysis were frozen at -80°C until processing.

Histological examination
The 10% formalin-fixed biopsy specimens were embedded in paraffin, and then the tissue sections were prepared for histopathological examinations.Classification and grading of gastric cancer were done using the Sydney system (Dixon et al., 1996).

DNA extraction
First, the biopsy specimens were introduced with a sterile needle on the slides and crushed by other slides carefully.The DNA extraction was then performed from all specimens according to the protocol for extraction by DNGTM-Plus kit (CinnaGen, Iran).Briefly, specimens were washed into tubes with 400 or 500 microliters (depending on sample size) of DNGTM-Plus solution.The tubes were vortexed until a completely homogenous suspension was obtained.Next, 300 microliters of isopropanol was added and vortexed for 3-5 second ,and held at -20°C temperature for 20 min.Tubes were centrifuged for 10 min at 12000rpm (revolutions per minute).The supernatants were discarded.The precipitates were re-suspended in 1 ml of 75% Ethanol and vortexed slowly and then centrifuged for 5 min at 12000rpm.The ethanol was poured off completely.This step was repeated once more.The pellet was dried at 65°C for 5min.DNA pellet was dissolved in 50 microliters of sterile distilled water by gentle shaking and placed at 65°C for 5min.Any residual pellets on the tube walls were dissolved by softly pippeting with distilled water.Finally, through a 30sec centrifuge at 12000rpm, the supernatant containing purified DNA was removed.

Primers and PCR conditions
In this study, genotyping of the 16S rDNA gene (for the confirmation of H pylori isolates) (Engstrand, 1992) and the vacA d region were determined by PCR methods.All primer sets were selected from published literature (Lu et al., 2002;Ogiwara et al., 2009).The PCR assay was performed in a reaction volume of 25μL using a commercially available kit (CinnaGen, Iran).The PCR conditions for vacAd1 and 16S rDNA were 95°C for 5min followed by thirty-seven cycles at 95°C for 45 s, 53°C for 60 s, and 72°C for 30 s; these 37 cycles were followed by one cycle at 72°C for 5 min.Finally, PCR products were separated by gel electrophoresis in 1.2% agarose gel, stained with ethidium bromide, and visualized on a UV transilluminator.Strains J99 and Tx30a were used as a positive control for d1 and d2 alleles respectively.

Statistical analysis
All data were analysed using SPSS version 19.The chi-square and Fisher's exact tests were used to evaluate the relationship between the frequency of each allele with the risk of gastric cancer and other gastroduodenal diseases.
The multiple linear regression analysis, after controlling for age and sex variables, was carried out to examine which allele (s) was related to gastroduodenal diseases.A stepwise method was used and variables were selected by the f value out and f value, F and f values were 3.84 and 0.05, respectively.
Logistic regression analysis was used to investigate the effect of each allele in gastric adenocarcinoma and gastroduodenal diseases, and the selection of variables was by the Enter method.A p value of less than 0.05 indicated significance.In all comparative analysis, patients with gastritis were considered as controls.

Presence of H pylori in gastric biopsy specimens and classification of patients
None of the test methods for detecting H pylori infection has been entirely ideal.Therefore, in addition to PCR amplification of H pylori 16S rDNA, rapid urease test or histological examination was used to confirm the presence of H pylori in biopsy specimens.
Eighty three patients tested positive for H pylori.Of these, 51 were classified as gastritis, 14 as peptic ulcer, and 18 as gastric adenocarcinoma.In this study, approximately 72% of patients were infected with H pylori and of these, 60% had gastritis.

Prevalence of H pylori vacA d1/-d2 genotypes and its influence on clinical outcome
This study showed that the frequencies of the vacA in d1, d2 were 43.4% and 56.6%, respectively.The frequency of the allele d1 (Table 3) was significantly higher in H pylori isolates from patients with gastric adenocarcinoma (66.6%) and peptic ulcer (71.4%) than in those with gastritis (27.4%).
The results of multiple linear/logistic regression analysis confirmed the high association of the vacA d1 genotype with gastric adenocarcinoma and Peptic ulcer rather than with gastritis.

Discussion
Many reports have shown an association between H pylori infection and the development of gastroduodenal diseases (Suerbaum and Michetti, 2002).However, the incidence of gastric cancer is enigmatically low in Africa and South Asia, where frequency of H pylori infection is high.Furthermore, several studies from East Asia and Iran have indicated a tendency towards reduction of GC incidence from north to south within these areas (Suzuki et al., 2012;Latifi-Navid et al., 2013).H pylori virulence factors have been shown to play an important role in determining the course of H pylori associated disease (Suerbaum and Michetti, 2002).Strains with the particular vacA genotypes have been found to be good markers for gastroduodenal disease (Atherton et al., 1995).Ogiwara et al. (2009) showed that the vacAd1 allele could be considered a more sensitive independent biomarker in the development of gastroduodenal disease, and Latifi-Navid et al. ( 2013) have also recently reported that the vacA d1/-i1 alleles from strains with European ancestry could be considered good markers for gastric cancer in Ardabil and Mazanderan (high incidence areas of GC in Iran) (Ogiwara et al., 2009;Latifi-Navid et al., 2013).In the present study, the genotype of the vacA d region was evaluated in H pylori isolated from patients with gastric adenocarcinoma, PUD and gastritis (control group) in East Azerbaijan in the Northwest of Iran.First, the frequency of vacA d1 was determined and compared with that reported in two previous studies (Ogiwara et al., 2009;Latifi-Navid et al., 2013).In this study, the frequencies for the vacA d1 and d2 alleles were 43.37% and 56/63% respectively in patients with gastric adenocarcinoma, peptic ulcer and gastritis.The prevalence of d1 allele is almost similar    to the pattern observed in a previous study (d1=39.9%)(Latifi-Navid et al., 2013).However, it is different from studies from western countries (d1=74.1%)and East Asian countries where almost 100% of the strains carry the vacA d1 genotype regardless of clinical outcomes (Ogiwara et al., 2009).In this study, the relationship between the vacA d region and different gastroduodenal diseases was also investigated.In agreement with findings from western countries, the strains carrying the vacA d1 genotype are significantly associated with gastric adenocarcinoma (66.6%) and peptic ulcer (71.4%), and these results were confirmed by multiple linear/logistic regression analysis (Ogiwara et al., 2009).In contrast, there is no significant correlation between the vacA genotypes and gastroduodenal disease in East Asian countries.The results of several studies have indicated that some Asian strains are influenced by genetic exchange with neighboring countries and are similar to other isolates from Western countries (Yamaoka et al., 2002;Azuma et al., 2004;Yamazaki et al., 2005;Satomi et al., 2006;Latifi-Navid et al., 2010), Whereas none of the Western strains have an East-Asian-type gene sequence (Xia et al., 2009).Since East Azerbaijan, like Ardabil and Mazanderan could be considered as the high incidence areas of GC in Iran (Somi et al., 2008;Latifi-Navid et al., 2013), H pylori strains from this region also might be similar to other isolates from Western Eurasia and placed in the hpEurope population (Latifi-Navid et al., 2010).Coleman et al. showed that almost 90% of cases of gastric cancer are adenocarcinomas (Coleman et al., 1993).In the present study, among the 34 gastric cancer patients, 27 (nearly 80%) had adenocarcinoma.
In agreement with other studies, the PCR technique possessed high sensitivity and specificity for the detection of H pylori in this study (Weiss et al., 1994).Sharp bands in PCR products of samples collected from urease test suggest that PCR is useful for diagnosing H pylori in gastric biopsy specimens.
In agreement with previous studies (Ogiwara et al., 2009;Latifi-Navid et al., 2013) we have proposed that the H pylori vacA d1 genotype might be a new risk marker for gastric adenocarcinoma and peptic ulcer in the Northwestern region of Iran.Regarding the strains carrying the virulence factors, vacA and cagA are particularly associated with the risk of gastric cancer (Huang et al., 2003;Atherton, 2006).We suggest that future studies on CagA EPIYA polymorphisms will also be required in these regions.We selected samples with sufficient size in this study.However, this study might be limited due to the number of samples collected.Future studies with a sufficient number of samples will also be required.
anti-inflammatory drugs orantihelicobacter therapy at least 3 months prior to endoscopy 41 Had a GI bleed 1 Had a previous history of peptic ulcer disease or gastric cancer 1* Had undergone a previous endoscopy or gastric surgery 0 Patients remaining in the study 115*This patient had also received nonsteroidal anti-inflammatoy drug