Jing Shen, Yao-Chu Xu, Xin-Ru Wang, Department of Epidemiology & Biostatistics, School of Public Health, Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
Run-Tian Wang, Health Science Center, Peking University, Beijing 100083, China
Li-Wei Wang, Yangzhong Cancer Research Institute, Yangzhong 212200, Jiangsu Province, China
Supported by Grants From the National Natural Science Foundation of China (30170827 to Jing. Shen and 30070671 to Run-Tian Wang)
Correspondence to: Jing Shen, Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, 701 West 168th St (Room 505), New York, NY 10032, USA.  js2182@columbia.edu
Telephone: +212-305-8158    Fax: +212-305-5328
Received: 2003-07-12    Accepted: 2003-10-12

Abstract
AIM: Inducible nitric oxide synthase (iNOS) plays a central role in the pathway of reactive oxygen and nitrogen species metabolism when Helicobacter pylori (H pylori) infection occurs in humans. iNOS Ser⁶⁰⁸Leu allele, a novel genetic polymorphism (C/T) occurring within exon 16 of the iNOS reductase domain, may have a dramatic effect on the enzymatic activity. The aim of this study was to determine whether iNOS C/T polymorphism was associated with increased susceptibility to gastric cancer.

METHODS: We conducted a population based case-control study in a high gastric cancer incidence area, Yangzhong, China. Questionnaires from 93 patients with intestinal type gastric cancer (IGC), 50 with gastric cardia cancer (GCC) and 246 healthy controls were obtained between 1997 and 1998, and iNOS genotyping was carried out. Odds ratios (ORs), interaction index (g), and 95% confidence intervals for the combined effects of iNOS genotype and H pylori infection, cigarette smoking or alcohol drinking were estimated.

RESULTS: The frequency of (CT+TT) genotypes was higher in cases than in control group (24.48% vs 23.17%), but the difference was not statistically significant. After adjusting for age and gender, past cigarette smokers with (CT+TT) genotypes had a significantly increased risk of IGC (OR = 3.62, 95% CI: 1.23-10.64), while past alcohol drinkers with (CT+TT) genotypes had a significantly increased risk of GCC (OR = 3.33, 95% CI: 1.14-9.67). H pylori CagA negative subjects with (CT+TT) genotypes had a significantly increased risk of both IGC and GCC (OR = 2.19 and 3.52, respectively).

CONCLUSION: iNOS Ser⁶⁰⁸Leu allele may be a potential determinant of susceptibility to cigarette -alcohol induced gastric cancer, but larger studies are needed to confirm the observations.

Shen J, Wang RT, Wang LW, Xu YC, Wang XR. A novel genetic polymorphism of inducible nitric oxide synthase is associated with an increased risk of gastric cancer. World J Gastroenterol  2004; 10(22): 3278-3283
http://www.wjgnet.com/1007-9327/10/3278.asp

INTRODUCTION
On a global scale, gastric cancer remains the world’s second most common malignancy. There is a substantial international variation in gastric cancer incidence with the highest rates reported from China, Japan and other Eastern Asian countries^[1]. The discovery of Helicobacter pylori (H pylori) in the early 1980 s has been proven to be a turning point in understanding the pathogenesis of this malignancy. A major advance in this field came with the recognition that chronic H pylori infection could induce physiologic and morphologic changes within the gastric milieu, which increase the risk of neoplastic transformation^[2]. It has been widely accepted that chronic H pylori infection induces hypochlorhydria and gastric atrophy, both of which are precursors of gastric cancer^[2]. Epidemiological studies have also indicated that infection with H pylori is considered as a risk factor for gastric cancer^[3,4] and the WHO IARC has classified this bacterium as a definite biological carcinogen^[5]. However, while the majority of infected individuals develop no significant clinical disease, others develop two kinds of divergent clinical outcomes-peptic ulcer disease and gastric cancer^[2]. The reasons for developing these two extreme phenotypes, especially important in gastric cancer, have remained poorly understood, and are not explained by bacterial virulence factors alone^[2]. This highlights the need to explore potential candidate genes of the host in the pathways involved in the natural history of H pylori infection and its interactions with other risk factors in the development of gastric cancer in a high-risk population.
      The inducible form of nitric oxide (NO) synthase (iNOS) is one of the most important enzymes involved in the pathway of reactive oxygen and nitrogen species metabolism in the presence of H pylori infection in humans. iNOS is a major source of NO production that is produced during inflammation by macrophages^[6,7]. Expression of iNOS in response to cytokines is part of the inflammatory response and contributes to tissue damage, suggesting its possible role in the processing of carcinogens^[7]. iNOS contains many sites for prosthetic groups and substrate binding^[8], which are all potentially important for the function of the enzyme. Furthermore, studies have indicated that even single amino acid changes may have dramatic effects on enzymatic activity^[8,9]. The human iNOS gene comprises 27 exons with the transcription start site in exon 2 (E2) and the stop codon in E27^[10]. E1-13 code for the oxygenase domain, and E1427 encode for the reductase domain of the protein. Both of the domains represent different functional parts of the enzyme^[9]. Increased iNOS activities have been observed in patients with chronic gastritis caused by H pylori infection, and gastric cancer^[11,12]. A 13-21 years follow-up study showed that among the H pylori positive group, the expression of iNOS and nitro-tyrosine was significantly higher in the group that developed gastric cancer than the one that showed no evidence of gastric cancer, suggesting that H pylori positive subjects with high levels of reactive nitrogen species in gastric mucosa may be a high-risk group for gastric cancer^[13]. Furthermore, recent studies have revealed that H pylori infection may lead to a sustained production of reactive nitrogen species and the formation of nitro-tyrosine contributes to DNA damage and apoptosis in gastric mucosa^[12]. Infection with H pylori strains possessing cytotoxin-associated gene (Cag) A, a molecular marker of H pylori virulence^[14,15], is particularly associated with an increased risk of developing adenocarcinoma of the stomach. It is suggested that iNOS may be a susceptible gene involved in the metabolic pathway of nitrogen and oxygen species of free radicals, and thus may be associated with both gastric cancer risk and H pylori infection.
       Yangzhong city is one of the areas in China with the highest gastric cancer mortality and incidence rate. The crude mortality rate of gastric cancer was from 96.9 to 110.9/100 000 during 1991 and 1997, and the average adjusted incidence rate in the same period was over 115/100 000 (unadjusted rate was 155.46/100 000), which is over ten times higher than that in the United States^[16]. Based on the understanding of the physiology and pathogenesis of gastric cancer, and the genetic pathway related to H pylori infection, we hypothesized that higher frequency of iNOS Ser⁶⁰⁸Leu allele (i.e. C/T polymorphism)^[9,17] was responsible for the higher gastric cancer incidence in this area. We were especially interested in knowing whether the association between the polymorphism and gastric cancer was modified by infection with H pylori CagA strains and cigarette smoking reflecting high exposure to nitrogen and oxygen species of free radicals. Therefore, the aim of this study was to determine whether iNOS C/T polymorphism was associated with increased susceptibility to gastric cancer, and the effects of H pylori infection.

MATERIALS AND METHODS
Study subjects
All gastric cancer patients and “healthy” controls in this study were Han ethnic Chinese living in Yangzhong city for at least 25 years. Gastric cancer was diagnosed according to the International Classification of Diseases for Oncology IX, code = 151, and the criteria of Laurén^[18]. Because most diagnosed gastric cancer cases in Yangzhong were intestinal type gastric cancer (IGC) and gastric cardia cancer (GCC), we focused on these two kinds of cancers in the present study. A population based case-control design was used, and 165 gastric cancer cases (108 IGC, 57 GCC) and 295 controls were enrolled. The finally analyzed cases and controls were 143 (93 IGC, 50 GCC) and 246 cases, respectively, because of missing genotype data for some subjects. There were no significant differences comparing the finally analyzed subjects and those with missing data by age and sex. All cases were identified by endoscopic and pathological diagnosis in Yangzhong City Municipal Hospital from January 1997 to December 1998. To reduce misclassification of the histological types, two pathologists reviewed and confirmed all diagnosed cases. Controls were selected from cancer-free subjects living in the same community, who were either cases siblings or their non-blood relatives (spouses and spouses siblings with the same gender as cases). Both types of controls differed slightly in demographic features^[19]. Their results were combined to increase the sample size and to decrease type I error. This study was approved by the regional ethics committee, and all participants were given an explanation of the nature of the study, and informed consents both written and oral, were obtained. Study subjects completed a questionnaire administered by trained interviewers.
     The questionnaire was designed to obtain detailed information on cigarette smoking, alcohol drinking, family history of cancers, and occupational and hazard exposures. Cigarette smokers were defined as subjects who reported ever smoking at least one cigarette per day for 12 mo or more, or whose accumulated cigarette consumption was over 18 packs per year. Past smokers were those who had stopped smoking 1 or more years before the interview. Alcohol drinkers were defined as subjects who reported to have an average of one drink or more per week for one or more yeaes. Past alcohol drinkers were also defined as those who had stopped drinking for 1 or more years before the interview.

Laboratory analysis
Blood was drawn from each participant by the designated coordinator according to the Guidelines of the National Heart, Lung, and Blood Institute Working Group on Blood Drawing, Processing, and Storage for Genetic Studies. Twenty milliliters of forearm venous blood was collected from each subject via venipuncture into two 10-mL vacutainer tubes containing EDTA. Puragene DNA isolation kits (Gentra Systems, Minneapolis MN) were used to isolate genomic DNA for genotyping. All blood samples were separated, and plasma was collected as soon as possible. The plasma was then stored at -20 °C in six 1.5-mL tubes for the detection of IgG antibody to H pylori CagA.
     Denaturing high performance liquid chromatography (DHPLC) was used to scan the potential single nucleotide polymorphisms (SNPs) in all exons of iNOS, and then sequencing was performed to confirm the possible mutations. Finally, a new C/T polymorphism, which changes the coding amino acid from serine (TCG) to leucine (TTG), was identified^[17]. PCR-RFLP was carried out to identify the genotype of iNOS according to the features of SNP, which created a restriction enzyme recognition site of Tsp 509 I. Genomic DNA was amplified with primers F: 5’-TGTAAACCAACTTCC GTGGTG-3’ (Tm =  60.82 °C) and R:  5’-GTCTCTGCGGGTCTGAGAAG-3’ (Tm = 60.14 °C). PCR was performed in a MJRESEARCH PCR system (PTC-225, USA), and in a 10 mL reaction volume containing 1mL 10×PCR buffer, 1.6 mL dNTPs (1.25 mmol/L), 0.2 mL MgCl₂ (25 mmol/L), primers (20 mmol/L, Resgen Corp.) at 0.15 mL each, DMSO 0.5 mL, 6.34 mL dH2O, 50 ng genomic DNA dried on the plate, and Hot Start Taq DNA polymerase 0.06 mL (5 U/mL, Promega Corp.). Touch down PCR procedure was used to amplify the target fragment. After an initial denaturation at 94 °C for 15 min, amplification was carried out for 10 cycles at 94 °C for 30 s, at 61 °C for 45 s, at 72 °C for 45 s and decreasing 0.5 °C per cycle. Then amplification was again carried out for 35 cycles at 94 °C for 30 s, at 56 °C for 45 s, and at 72 °C for 45 s, followed by a final elongation at 72 °C for 7 min. Then, 10 mL PCR products was digested with 0.2 mL Tsp 509 I (10 U/mL, NEB Corp.) in a 15 mL volume including 2 mL 10×buffer 1 (NEB Corp.), 0.15 mL BSA (100×) and 2.65 mL dH2O. Digestion was performed for 15 h at 65 °C. The products were then electrophoresed on a 30 g/L agarose gel to allow unambiguous detection with ethidium bromide staining. Homozygous wide-type individuals (CC) showed 113 bp and 175 bp fragments, heterozygous individuals (CT) showed three bands: 113 bp, 142 bp and 175 bp, and homozygous rare allele individuals (TT) showed two bands: 113 bp and 142 bp^[17].
      H pylori CagA IgG antibody in plasma was measured by an enzyme-linked immunosorbent assay (ELISA) kit offered by Jingying Biotech Limited Company, Shanghai, China (batch number 0052). The Absorbency at 450 nm was determined after terminating the enzyme reaction. The cutoff value equaled to the average A of the negative controls provided by the manufacturer plus 0.3 A units. A values of samples equaled to or higher than the cutoff point were considered positive.

Statistical analysis
All data were input double blinded into EPI-6 program by two persons separately. After modifying all errors and non-logical data, the differences in the relative associations between cases and controls were assessed by calculating crude odds ratios (OR) from contingency tables. The corresponding chi-square test on the cancer patients and controls was carried out, and 95% confidence intervals (95% CI) were determined using the Fisher exact test. A P-value <0.05 was considered statistically significant. Unconditional logistic regression analysis was performed in both univariate and multivariate models to assess the association between iNOS functional polymorphism and gastric cancer susceptibility after adjusting for important confounding factors such as age and sex. Test of trend and interaction index (g) that was determined by coefficient (b) in a multiple logistic regression model were calculated through logistic models based on dummy variables to examine the potential gene-environment interaction^[20]. All analyses were performed with the SAS package Genmod (SAS Institute, Cary, NC).

RESULTS
Table 1 compares the characteristics of study subjects. The mean age of cases was significantly greater than the controls (59.36 vs 51.89, P<0.01). There was no significant difference in the male/female ratio between cases and controls. The proportions of past smokers and alcohol drinkers was significantly greater in the cancer group (36.97% and 30.30%) than in the control group (12.54% and 14.92%). However, there were more current smokers and drinkers in the control (47.12% and 30.85%) than in the case group (26.67% and 10.91%). Compared with controls, cases were significantly less likely to be positive for H pylori CagA antibody.
      The frequency of iNOS genotypes in gastric cancer and control subjects showed no significant difference, although the frequency of (CT+TT ) genotypes was slightly higher in cases than in controls (24.48% versus 23.17%). A gene dose-response effect was not observed, i.e. the effect of heterozygote (CT) genotypes did not lie at or between the homozygotes (CC and TT).
      For (CT+TT) the genotype frequency of iNOS in the past smoking subgroup, there were significant differences between the total cases and the controls and between GCC group and controls with an OR of 3.62 (95% CI: 1.23-10.64) and 4.63 (95% CI: 1.15-18.58), respectively. No significant difference was found between IGC cases and controls (Table 2). Although no gene dose-response effect was observed in heterozygote (CT) and homozygote (TT) individuals because of the small number in each cell, there was still a possible interaction between C/T polymorphism and past cigarette smoking in increasing the risk of GCC. In the past alcohol drinkers, there were significant differences in the C/T polymorphism between total cases and controls and between IGC group and controls with an OR of 3.33 (95% CI: 1.14-9.67) and 3.42 (95% CI: 1.03-11.35), respectively. No significant difference between GCC group and control group was found (Table 3). In H pylori CagA negative group, subjects with (CT+TT) genotypes had significantly increased risk of both IGC and GCC, with an OR of 2.19 (95% CI: 1.01-4.76) and 3.52 (95% CI: 1.44-8.61), respectively. H pylori CagA positivity showed significant protective effects in IGC group in both on CC and CT+TT iNOS genotypes, with an OR of 0.24 (95% CI: 0.07-0.89) and 0.34 (95% CI: 0.17-0.70), respectively. However, no significant association was observed between iNOS genotypes and  GCC (Table 4).

Table 1  Covariate distribution among study subjects and ORs for gastric cancer

^aP<0.05 vs control group after adjusted for age and gender, ^bP<0.01 vs control.

Table 2  Interaction between C/T polymorphism and past cigarette smoking for the risk of gastric cancer

¹Adjusted for age and gender, x²_trend = 26.26, ^df = 1, P = 0.00, g = 1.29/1.07 = 1.21 ²Adjusted for age and gender, x²_trend = 18.40, ^df = 1, P = 0.00, g = 1.12/0.90 = 1.24 ³Adjusted for age and gender, x² _trend = 17.53, ^df = 1, P = 0.00, g = 1.53/1.29 = 1.19.

Table 3  Interaction between C/T polymorphism and past alcohol drinking for the risk of gastric cancer

¹Adjusted for age and gender, x²_trend = 10.29, ^df = 1, P = 0.001, g = 1.20/0.30 = 4.00 ²Adjusted for age and gender, x²_trend = 5.65, ^df = 1, P = 0.017, g = 1.23/0.31 = 3.97 ³Adjusted for age and gender, x²_trend = 8.95, ^df = 1, P = 0.003, g = 1.18/0.24 = 4.9.

Table 4  Interaction between C/T polymorphism and H pylori CagA status for the risk of gastric cancer

¹Adjusted for age and gender, x²_trend = 33.40, df = 1, P = 0.00, g = -0.84/-0.79 = 1.06 ²Adjusted for age and gender, x²_trend = 28.53, ^df = 1, P = 0.00, g = -1.41/-1.08 = 1.31 ³Adjusted for age and gender, x²_trend = 12.42, ^df = 1, P = 0.0004, g = -0.15/-0.27 = 0.56.

DISCUSSION
H pylori infection could produce a state of chronic immuno-stimulation in gastric epithelium^[21]. It could lead to changes in many factors that are important in the pathogenesis of gastric cancer, including reactive oxygen and nitrogen oxide species ^[22]. NO, a potentially toxic gas with free radical properties is one of the most important bio-regulatory and signaling molecules produced in the process. It has been recently reported that NO, acting as a messenger molecule mediating various physiological functions^[23,24], may also play a role in the process of carcinogenesis.
      It has been found that NO is synthesized enzymatically from L-arginine by NO synthase^[23,25]. Chronic infection and immuno-stimulation elevate endogenous synthesis of NO. High concentration of NO generated by macrophages after iNOS induction contributed to their cytotoxic and carcinogenic activity^[26]. There is now increasing evidence that NO produced by activated phagocytes may play a role in multistage carcinogenesis by mediating DNA damage^[27,28]. A to T substitution in the iNOS gene, leads to more activated iNOS expression in the target cells, and finally elevates NO to a high level. Hence, it is reasonable to assume that human iNOS gene may be another important candidate gene for the development of gastric cancer by elevating NO production in target cells when functional polymorphisms occur. Nevertheless its genomic localization at chromosome 17q11.2^[29] was not the same as other gastric cancer susceptible genes related to the inflammatory response pathway, such as interleukin 1b and interleukin 1RN, located at 2q14^[30]. The key question for gastric cancer agents is how H pylori infection could be associated with such totally divergent clinical outcomes as gastric cancer and peptic ulcer disease. A large number of previous studies have focused on the role of the bacterial virulence factors that contribute to the degree of tissue damage in the pathogenesis of these diseases. But these results still could not explain the different outcomes^[2,22,31]. With the development of a key concept about the interaction between acid secretion and H pylori-induced gastritis during 1990 s, El-Omar proposed the idea for the first time that host genetic factors relevant to pro-inflammatory responses might be relevant to the development of gastric cancer. They explored a candidate IL-1b gene in the context of H pylori related disease^[32,33]. Because IL-1b can also induce the expression of many other genes, including pro-inflammatory mediator iNOS, by either regulating at the transcriptional level or initiating their mRNA^[8,9,34], it is easy to consider that functional polymorphisms occurring in the iNOS gene might also contribute to the increased risk of H pylori related gastric cancer.
     We have previously reported a newly discovered C/T polymorphism in a Chinese population^[17] that had a high mutated allele frequency (24.4%). A report by Johannesen also showed that C/T polymorphism was one of the most frequent SNPs among 10 polymorphisms of human iNOS gene identified in a Danish population. They suggested that the amino acid change in exon 16 might be of functional interest^[9]. Our results showed no significant difference in the frequency of (CT+TT) genotypes between cases and controls, and no apparent gene dose-response effect was found. However, in past cigarette smokers and past alcohol drinkers, C/T polymorphism significantly increased the risk of gastric cancer despite the histological subtypes differed, i.e. past cigarette smokers with (CT+TT) genotypes had an increased risk of IGC, while past alcohol drinkers with (CT+TT) genotypes had increased risk of GCC. These findings suggest that C/T polymorphism in iNOS gene alone is not sufficient to show the increasing risk of gastric cancer. The importance of the interaction between C/T polymorphism and cigarette smoking or alcohol drinking varied depending on different histological subtypes of gastric cancer. Similar results were found by Machado for IL-1 genetic markers^[35]. Although these findings were not the major hypothesis we proposed, it is biologically plausible that oxidative stress due to carcinogenesis in cigarette might attribute to the increase of gastric cancer risk through interactions with iNOS C/T polymorphism. Larger and independent studies are needed to confirm these findings.
      In the H pylori CagA positive group, regardless of whether subjects had CC or (CT+TT) genotypes, we always observed a significant protective effect when comparing IGC cases with controls. This suggests that plasma positive for H pylori CagA antibody in a highly infected area plays a protective role. This is in concordance with the finding that H pylori density became progressively lower with progression from mild gastritis to severe gastritis, atrophy, intestinal metaplasia and finally gastric cancer^[29]. In H pylori CagA negative subjects with (CT+TT) genotypes, a high risk was found for gastric cancer group (OR = 2.53, 95% CI: 1.29-4.98) and both subgroups (IGC and GCC). No interaction was found between iNOS genotype and infection with H pylori CagA strains.
      iNOS protein is a catalytic enzyme with two domains. In terms of functional importance, the deletion mutants retained maximal NO activity at lower concentrations of free Ca²⁺ compared with the wild-type^[36]. Identified C/T polymorphism in E16 of iNOS was located at the N-terminal of six amino acids from the deletion reported by Daff et al., and the amino acid change in E16 might be of functional interest^[9]. To our knowledge, this study was the first one to examine the significance of iNOS polymorphism in gastric cancer. A research on other type of disease might support our observation^[9]. Gastric cancer patients having allele T polymorphism could have an increased expression of iNOS, resulting in higher levels of NO in gastric mucosa, mediating many pathological changes and finally leading to carcinogenesis in these patients. But specific functional tests of C/T shift need to be performed to substantiate the putative importance of the Ser608Leu locus in gastric cancer development.
     Potential weaknesses in our study include possible recruitment bias in the selection of controls including cases siblings. This kind of selection might create overmatching. Siblings were more likely to have the same genotypes as the cases than the non-blood related controls, thereby leading to some loss of statistical efficiency, i.e., larger sample sizes were required to attain the same statistical precision^[37]. Thus, our data may be more likely to underestimate the true effect of iNOS T alleles on the risk of gastric cancer. But others considered that the use of sibling controls could generally improve efficiency for gene-environment interactions^[38,39]. We could not rule out the potential influence of systematic differences between participants and non-participants.
      In conclusion, the risk of gastric cancer is increast among past cigarette smoking or alcohol drinking individuals with a C/T polymorphism in E16 of iNOS gene in a Chinese population. But the findings need to be confirmed in other ethnic populations.

ACKNOWLEDGEMENTS
We thank Zhao-Xi Wang for his excellent technical assistance; and Professor Regina M. Santella and Dr. Yu-Jing Zhang for editing the manuscript. We also thank all the doctors for their kind help in collecting the biological samples and epidemiological data. We thank all participatants for their co-operation.

REFERENCES
1    Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide incidence of 25 major cancers in 1990. Int J Cancer 
      1999; 80: 827-841
2    El-Omar EM, Chow WH, Rabkin CS. Gastric cancer and H pylori: Host genetics open the way. Gastroenterology 
      2001; 121:1002-1004
3    Correa P. Human gastric carcinogenesis: a multistep and multifactorial process-first american cancer society award 
      lecture on cancer epidemiology and prevention. Cancer Res 1992; 52:6735-6740
4    Komoto K, Haruma K, Kamada T, Tanaka S, Yoshihara M, Sumii K, Kajiyama G, Talley NJ. Helicobacter pylori infection 
      and gastric neoplasia: correlations with histological gastritis and tumor histology. Am J Gastroenterol 
      1998; 93: 1271-1276
5    Schistosomes, liver flukes and Helicobacter pylori. IARC Working Group on the Evaluation of Carcinogenic Risks to 
      Humans. Lyon, 7-14 June 1994. IARC Monogr Eval Carcinog Risks Hum 1994; 61: 1-241
6    Felley CP, Pignatelli B, Van Melle GD, Crabtree JE, Stolte M, Diezi J, Corthesy-Theulaz I, Michetti P, Bancel B, Patricot LM, 
      Ohshima H, Felley-Bosco E. Oxidative stress in gastric mucosa of asymptomatic humans infected with Helicobacter 
      pylori: effect of bacterial eradication. Helicobacter 2002; 7: 342-348
7    Vallance P, Collier J. Biology and clinical relevance of nitric oxide. Br Med J 1994; 309: 453-457
8    Stuehr DJ. Mammalian nitric oxide synthases. Biochim Biophys Acta 1999; 1411: 217-230
9    Johannesen J, Pie A, Paiot F, Kristiansen OP, Karlsen AE, Nerup J. Linkage of the human inducible nitric oxide synthase 
      gene to type 1 diabetes. J Clin Endocrinol Metab 2001; 86: 2792-2796
10  Xu W, Charles IG, Liu L, Moncada S, Emson P. Molecular cloning and structural organization of the human inducible 
      nitric oxide synthase gene (NOS2). Biochem Biophys Res Commun 1996; 219: 784-788
11  Mannick EE, Bravo LE, Zarama G, Realpe JL, Zhang XJ, Ruiz B, Fontham ET, Mera R, Miller MJ, Correa P. Inducible nitric 
      oxide synthase, nitrotyrosine, and apoptosis in Helicobacter pylori gastritis: effect of antibiotics and antioxidants. Cancer 
      Res 1996; 56: 3238-3243
12  Wee A, Kang JY, Teh M. Helicobacter pylori and gastric cancer: correlation with gastritis, intestinal metaplasia, and 
      tumour histology. Gut 1992; 33: 1029-1032
13  Rajnakova A, Goh PM, Chan ST, Ngoi SS, Alponat A, Moochhala S. Expression of differential nitric oxide synthase 
      isoforms in human normal gastric mucosa and gastric cancer tissue. Carcinogenesis 1997; 18: 1841-1845
14  Blaser MJ, Perez-Perez GI, Kleanthous H, Cover TL, Peek RM, Chyou PH, Stemmermann GN, Nomura A. Infection with 
      Helicobacter pylori strains possessing CagA is associated with an increased risk of developing adenocarcinoma of the 
      stomach. Cancer Res 1995; 55: 2111-2115
15  Goto T, Haruma K, Kitadai Y, Ito M, Yoshihara M, Sumii K, Hayakawa N, Kajiyama G. Enhanced expression of inducible 
      nitric oxide synthase and nitrotyrosine in gastric mucosa of gastric cancer patients. Clin Cancer Res 1999;5:1411-1415
16  Stadtlander CT, Waterbor JW. Molecular epidemiology, pathogenesis and prevention of gastric cancer. Carcinogenesis 
      1999; 20: 2195-2208
17  Shen J, Wang RT, Wand LW, Wang ZX, Xing HX, Wang BY, Guo CH, Wang XR, Xu XP. Ser/Leu polymorphism of iNOS 
      gene was found and identified in Chinese by denaturing high performance liquid chromatography. Peking Daxue Xuebao 
      2001; 33: 486-492
18  Laurén P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An 
      attempt at a histo-clinical classification. Acta Pathol Microbiol Scand 1965; 64: 31-49
19  Shen J, Wang R, Wang Z, Xing H, Wang L, Wang B, Li M, Hua Z, Wang J, Guo C, Wang X, Xu X. The distributive 
      features of three kinds of metabolic genes polymorphisms in population of Han nationality in south area of China. 
      Zhonghua Yixue Yichuanxue Zazhi 2002; 19: 302-307
20  Taioli E, Zocchetti C, Garte S. Models of interaction between metabolic genes and environmental exposure in cancer 
      susceptibility. Environ Health Perspect 1998; 106: 67-70
21  Shapiro KB, Hotchkiss JH. Induction of nitric oxide synthesis in murine macrophages by Helicobacter pylori. Cancer Lett 
      1996; 102: 49-56
22  El-Omar EM. The importance of interleukin 1 beta in Helicobacter pylori associated disease. Gut 2001; 48: 743-747
23  Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 
      1991; 43:109-142
24  Son HJ, Rhee JC, Park DI, Kim YH, Rhee PL, Koh KC, Paik SW, Choi KW, Kim JJ. Inducible nitric oxide synthase 
      expression in gastroduodenal diseases infected with Helicobacter pylori. Helicobacter 2001; 6: 37-43
25  Forstermann U, Schmidt HH, Pollock JS, Sheng H, Mitchell JA, Warner TD, Nakane M, Murad F. Isoforms of nitric oxide 
      synthase. Characterization and purification from different cell types. Biochem Pharmacol 1991; 42: 1849-1857
26  Grisham MB, Ware K, Gilleland HE Jr, Gilleland LB, Abell CL, Yamada T. Neutrophil-mediated nitrosamine formation: role 
      of nitric oxide in rats. Gastroenterology 1992; 103: 1260-1266
27  Esumi H, Tannenbaum SR. US-Japan Cooperative Cancer Research Program: seminar on nitric oxide synthase and 
      carcinogenesis. Cancer Res 1994; 54: 297-301
28  Ohshima H, Bartsch H. Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide 
      in carcinogenesis. Mutat Res 1994; 305: 253-264
29  Marsden PA, Heng HH, Duff CL, Shi XM, Tsui LC, Hall AV. Localization of the human gene for inducible nitric oxide 
      synthase (NOS2) to chromosome 17q11.2-q12. Genomics 1994; 19: 183-185
30  Patterson D, Jones C, Hart I, Bleskan J, Berger R, Geyer D, Eisenberg SP, Smith MF Jr, Arend WP. The human 
      interleukin-1 receptor antagonist (IL1RN) gene is located in the chromosome 2q14 region. Genomics 
      1993; 15: 173-176
31  Graham DY, Yamaoka Y. Disease-specific Helicobacter pylori virulence factors: the unfulfilled promise. Helicobacter 
      2000; 5(Suppl 1): S3-S9
32  El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, Herrera J, Lissowska J, Yuan CC, Rothman N, 
      Lanyon G, Martin M, Fraumeni JF Jr, Rabkin CS. Interleukin-1 polymorphisms associated with increased risk of gastric 
      cancer. Nature 2000; 404: 398-402
33  El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, Herrera J, Lissowska J, Yuan CC, Rothman N, 
      Lanyon G, Martin M, Fraumeni JF Jr, Rabkin CS. The role of interleukin-1 polymorphisms in the pathogenesis of gastric 
      cancer. Nature 2001; 412: 99
34  Cho HJ, Xie QW, Calaycay J, Mumford RA, Swiderek KM, Lee TD, Nathan C. Calmodulin is a subunit of nitric oxide 
      synthase from macrophages. J Exp Med 1992; 176: 599-604
35  Machado JC, Pharoah P, Sousa S, Carvalho R, Oliveira C, Figueiredo C, Amorim A, Seruca R, Caldas C, Carneiro F, 
      Sobrinho-Simoes M. Interleukin 1B and interleukin 1RN polymorphisms are associated with increased risk of gastric 
      carcinoma. Gastroenterology 2001; 121: 823-829
36  Daff S, Sagami I, Shimizu T. The 42-amino acid insert in the FMN domain of neuronal nitric-oxide synthase exerts 
      control over Ca(2+)/calmodulin-dependent electron transfer. J Biol Chem 1999; 274: 30589-30595
37  Thomas DC, Witte JS. Point: population stratification: a problem for case-control studies of candidate-gene 
      associations? Cancer Epidemiol Biomark Prev 2002; 11: 505-512
38  Gauderman WJ, Witte JS, Thomas DC. Family-based association studies. J Natl Cancer Inst Monogr 1999; 26: 31-37
39  Witte JS, Gauderman WJ, Thomas DC. Asymptotic bias and efficiency in case-control studies of candidate genes and 
      gene-environment interactions: basic family designs. Am J Epidemiol 1999; 149: 693-705

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