Study On Helicobacter Pylori HrgA Status And Preponderant Genes Of Zhejiang Province

Background and Aim Heliobacter pylori(H. pylori) is a spiral Gram-negative microaerophilic bacterium that chronically colonizes the gastric epitheliums of humans. H. pylori is identified as the main cause of chronic active gastritis and peptic ulcer diseases (PUD) in human beings and is considered to be a risk factor for the development of gastric mucosa-associated lymphoid tissue lymphoma. Accordingly, the World Health Organization has classified H. pylori as a group I carcinogen in 1994. The organism has a worldwide distribution and its prevalence ranges from 20-50% in developed countries to over 70% in developing areas. 20~30% of the total population in Chinese have chronic gastric disease and some of them develop into PUD or even gastric carcinoma. Certain nosogenesis about the different clinical outcomes is unknown yet. One explanation might be the diversified genetype of H. pylori strains, lots of epidemiologic research indicates that several putative bacterial virulence factors such as cagA, vacA, iceA and babA are closely associated with PUD and (or) gastric carcinoma. However, the conclusion is not completely coherent in different countries. Therefore, the study on virulence genes of H. pylori is still a hot issue.A recently identified gene of H. pylori, restriction endonuclease-replacing gene(hrgA) who replaced hpy\RR gene ,was reported to be precent in 70(34%) of 208 strains examined (Ando et al). Ando' s reseach indicated that hrgA was a strain-specific gene that might be associated with gastric cancer among H. pylori isolates from Asian patients and it might be used to identify individuals of high gastric cancer risk. No artical about hrgA gene is reported in China until now. Furthermore, many of studies proved that, cag^-positive H. pylori strains were highly associated with peptic ulcer disease and with the risk of developing intestinal metaplasia, atrophic gastritis and gastric carcinoma. VacA gene which encodes the vacuolating toxin, is present in all strains, and comprises 2 variable parts, s- and m-region. The mosaic combination of s- and m- region allelic types determines the quantity and activity of cytotoxin and is associated with pathogenicity of the bacterium. BabA is one of the best defined H. pylori adhesins found to date and it has been shown to mediate adherence of H. pylori to human Lewis b blood group antigen on gastric epithelial cells. IceA gene exists as two distinct genotypes, iceAl and iceA2. And in vivo carriage of H. pylori iceAl strains has been reported to be associated with peptic ulceration and enhanced acute neutrophilic infiltration. But there is no systemically study on all of genes above in China. Our study aim to find out whether the presence of hrgA has disease specificity, meanwhile, we observed cagA, vac A, ice A and bob A in Zhejiang Province and tried to find out the relationship between these genes and gastric mucous membrane diseases.Materials and Methods A total of 132 H. pylori isolates were obtained from H.pylori-in&cted adults living in Zhejiang Province. Sixty-nine isolates were from the patients in Hangzhou City and the others were from the patient in Daishan country .The patients which consisted of 89 men and 43 women with a mean age 45.6 years (ranging from 16 to 70 years), were classified into 3 groups including chronic gastritis (?=64), peptic ulcer (?=56) and gastric adenocarcinoma (?=12),according to the results of endoscopic and histological examinations. H. pylori isolates from biopsy specimens were cultured on ECY selective solid medium at 37°C for 5 days, under 98% humidity and microaerophilic conditions (5%C>2, 10%CO2, and 85%N2). H. pylori were identified by the following criteria: characteristic of colony, rapid urease test, catalase test and morphology on Gram staining. H. pylori isolates were harvested from the agar plates and genomic DNA was extracted by phenol/chloroform method. The primers were according to the references. The hrgA, cagA, vacA, iceAl and babAl genotypes were determined by PCR method. The reaction conditions were 3 minutes pregeneration at 95 °C, followed by 30 cycles of 45 seconds at 94°C, 45 seconds at 54°C, and 45 seconds at 72 °C. Final extension was performed for 7 minutes at 72 "C. The relationship between individual genotypes and the different gastric diseases was assessed with Chi-square (x2) test, a P value of <0.05 was defined as significant.Results and Discussion Every product of PCR was coincident to the references. Of 132 H. pylori isolates from Zhejiang population, 34(25.8%) yielded hrgA PCR product of the expected size (594bp). H. pylori possess either hpyWK or hrgA, nevertheless, in not one of the 132 strains tested were both genes missing or were both genes present. The prevalence of hrgA in chronic gastritis, PUD and gastric carcinoma is 23.4%, 28.6% and 25.0%, respectively. The prevalence of the hrgA gene was not related to clinical outcome. The cagA, vacAsla, vacAm2, iceAl, and babA2 genetypes were detected in 100%, 92.4%, 81.8%, 83.3% and 78.9%, respectively, of the H. pylori strains studied. There was no relationship between the prevalence of cagA, iceAl, babA2 and vacAsla genotypes and gastric duodenal diseases in Zhejiang province. In the patients with PUD, vacAm2 is markedly more than in those with chronic gastritis (P<0.01). VacAvcQ. genotype could serve as biomarker for the risk of PUD.Conclusion The prevalence of hrgA gene is 25.8% that is consistent with thereferences, and in all of the tested strains, there is no one strain whose hpyWR and hrgA genes are both missing or are both present. The prevalence of the hrgA gene is not related to clinical outcome .The cagA, vacAsla/m2, iceAl and babA2 genotypes H. pylori is predominant in Zhejiang Province. VacAml gene might be associated with PUD.