Lack of association of the iNOS gene polymorphism with risk of cancer: a systematic review and Meta-Analysis

In order to investigate the association between the iNOS gene polymorphisms and susceptibility to cancer, a search of English papers was done using Pubmed, the Cochrane Library, Embase, ISI Web of Science, Google (scholar) database, and all Chinese reports were conducted using CBMDisc, Chongqing VIP database, and CNKI database. A total of eight studies were included in this meta-analysis including 1,920 cases and 2,373 controls. The results indicated that the polymorphisms in iNOS gene (C150T(Ser608 Leu) polymorphism and polymorphic (CCTTT)n repeats) had no association with cancer risk for all genetic models. This meta-analysis suggested that the polymorphisms in the iNOS gene were not associated with cancer risk.

in Table 1 and Table 2. Genotype distribution of any polymorphism did not differ from HWE within control groups.
Sensitivity Analysis. In order to examine the influence of the individual data set to the pooled ORs, every single study was deleted each time. Sensitivity analysis indicated our results were statistically robust (data not shown).

Discussion
As a production of nitric oxide (NO), iNOS is produced during inflammation by macrophages 14 . Expression of iNOS in response to cytokines is one of the important sections for inflammatory reaction and relates to angiogenesis, suggesting its potential role in the process of carcinogens 15 . The polymorphism (C150T) in exon 16 of iNOS gene results in an amino acid substitute, Ser 608 Leu. The iNOS Ser 608 Leu allele (C > T polymorphism) leads an amino acid alteration in a regulatory domain of the enzyme 16 . Furthermore, a polymorphic pentanucleotide (CCTTT)n repeat probably 2.5 kilobase substream the transcription initiation site has been clarified to affect iNOS expression 17 . In our meta-analysis, the associations between these two polymorphisms in iNOS gene and risk of cancer were studied.
Except detection the expression of iNOS in vivo or in vitro with tumor 18,19 , there were some other researches in cancer patients that susceptibility to carcinogenesis may be correlated with the existence of particular alleles at the iNOS locus. However, the results are inconsistent and inconclusive due to limited sample size and different study methods. To the best of our knowledge, this is the first meta-analysis to evaluate iNOS gene polymorphism in development and growth of cancer. In order to acquire a more reliable and comprehensive evidence on both variants, we conducted this meta-analysis to evaluate the association between the polymorphism in iNOS gene and risk of cancer on the basis of data from eight studies. The results indicated that there was no significant association for iNOS gene polymorphism and risk of cancer. In addition, another iNOS gene (iNOS974) was studied in the relationship with cancer. However, there was no significant difference between iNOS974 polymorphism and cancer risk 9 .
Although we have not found a significant association between iNOS gene polymorphisms and cancer risk, some studies revealed that the risk of cancer is increasing among smoking or drinking individuals with polymorphism in iNOS gene 5,6 . In addition, studies also reported that iNOS gene polymorphism was associated with the risk of H pylori-related gastric cancer 3,8 . We could provide a hypothesis that there was an association between iNOS gene and risk of cancer combined with risk factors including lifestyle and H pylori infection. As the reason for heterogeneity (including different study methods and outcomes) among studies, we could not use meta-analysis to analyze the relationship between iNOS genes combined with those risk factors and cancer.
This meta-analysis has pooled the available data from the eligible studies, which has significantly increased the statistical power. Nevertheless, the results of the present meta-analysis should also be explained within the context of its limitations. First, cancer is a multi-factorial illness from complex interactions between environmental exposure and genetic factors. In this meta-analysis, we had insufficient data to conduct an evaluation of such interactions for the role of iNOS polymorphisms and factors in cancer development. Second, the number of current studies is relative limited. Thus, investigations involving large number of different races are necessary for a more reliable assessment on their associations. Third, our meta-analysis is based on unadjusted estimate because lacking of sufficient data. Forth, although we have made our best efforts to avoid all potential publications, it is likely that some are missed or displayed erroneously. Furthermore, we did not include studies published in language other than English or Chinese.
In conclusion, this meta-analysis revealed that the polymorphisms in iNOS gene could not be regarded as a strong genetic risk factor for cancer but it might be association with cancer combined with additional risk factors. In addition, this result should be interpreted cautiously. In order to better understand the potential etiology for cancer in human, large well-designed studies in the susceptibility of cancer evidence are needed to perform in future. It also will be necessary to combine genetic factors and other environmental risk factors.

Methods
Selection of eligible studies. Studies on the associations between iNOS gene polymorphisms and cancer were scrutinized by two reviewers (L.L. and J.H.J.) independently. We searched Pubmed, Embase, the Cochrane Library, ISI Web of Science, Google (scholar) database, Chinese Biological Medicine Disc (CBMDisc), China National Knowledge Infrastructure (CNKI), and Chongqing VIP database (Last search was updated on December 10, 2014) using the terms "inducible nitric oxide synthase or iNOS", "cancer or tumor or carcinoma" and "polymorphism, variant or mutation". Reference lists were checked and additional literatures were contacted by researchers. Authors of publications were contacted when results were unclear or when sufficient data were not reported. The search was performed without restriction on language, but we only included articles written in English or Chinese. Selection Criteria. Studies were included if they fulfilled all of the following entry criteria: (1) it must be a case-control or cohort study design; (2) there were sufficient data for iNOS gene mutations with risk of cancer; (3) the genotype distribution in the controls of all studies should be in agreement with Hardy-Weinberg equilibrium (HWE); and (4) in the case of multiple publications from the same study group, the most complete and recent results were used.

Publication
Year   Data extraction. After studies selection, two investigators independently extracted data from each study with a standard form and entered into a database. When discrepancies were appeared, all investigators were recruited to evaluate the data. The following information was collected: First author, publication year, location, ethnicity, characteristics, sample sizes of patients and controls, genotype numbers.  Table 3. Summary ORs and 95% CI of the C150T(Ser 608 Leu) Polymorphism and the polymorphic (CCTTT)n repeats in the inducible nitric oxide synthase Gene and Cancer Risk. OR: odds ratio; CI: confidence interval. *repeat numbers divided into two groups: S (9-11 repeats) and L (12-18 repeats).