We combined the outcomes of all randomised controlled trials to investigate the safety and efficacy of steroid avoidance or withdrawal (SAW) regimens in paediatric kidney transplantation compared with steroid-based (SB) regimens.A systematic literature search of PubMed, Embase, Cochrane Library, the trials registry and BIOSIS previews was performed. A change in the height standardised Z-score from baseline (ΔHSDS) and acute rejection were the primary endpoints.Eight reports from 5 randomised controlled trials were included, with a total of 528 patients. Sufficient evidence of a significant increase in the ΔHSDS was observed in the SAW group (mean difference (MD) = 0.38, 95% confidence interval (CI) 0.07–0.68, P = 0.01), particularly within the first year post-withdrawal  and in the prepubertal recipients . There was no significant difference in the risk of acute rejection between the groups .The SAW regimen is justified in select paediatric renal allograft recipients because it provides significant benefits in post-transplant growth within the first year post-withdrawal with minimal effects on the risk of acute rejection, graft function, and graft and patient survival within 3 years post-withdrawal. These select paediatric recipients should have the following characteristics: prepubertal; Caucasian; with primary disease not related to immunological factors; de novo kidney transplant recipient; with low panel reactive antibody. Steroids have been widely used in immunosuppressive regimens for paediatric kidney allograft recipients. However, the long-term administration of steroids leads to multiple adverse effects, even at a minimal dose. In paediatric recipients, steroid-induced growth retardation is of particular concern .Attempts have been made to avoid or withdraw steroid therapy in paediatric kidney allograft recipients. Several meta-analyses have evaluated the efficacy and safety of steroid avoidance or withdrawal (SAW) protocols in adult renal transplant recipients –7. The mth, 2015.RCTs comparing the beneficial and harmful effects of SAW regimens with those of steroid-based regimens in paediatric renal transplant recipients were included. The definition of a child varied among countries, and all definitions were accepted. A systematic literature search of PubMed, Embase, Cochrane Library and the trials registry was performed. Conference abstracts and proceedings were searched using BIOSIS previews. Searches were conducted using MeSH keywords and free-text aliases for corticosteroids, child, KTx and RCTs in each database . No langThe primary efficacy outcome was linear growth post-transplant, as indicated by a change in height standardised Z-score (ΔHSDS) from baseline. The primary safety outcome was AR. The secondary outcomes were patient and graft survival, renal graft function (expressed as the estimated glomerular filtration rate (eGFR)) and adverse events, including delayed graft function (DGF), hypertension, new-onset diabetes after transplant (NODAT), hyperlipidaemia, infection and post-transplant lymphoproliferative disorders (PTLDs). All outcomes were analysed at different time points.Studies were selected independently by two authors according to the inclusion criteria. The studies are referred to by the year of the latest reportand its first author. Data were extracted from the published reports and recorded on specific data collection forms by one author and were then verified by another author. Disagreements were resolved by discussion. Data from reports originating from the same clinical trials were combined for analysis. When data could not be pooled directly for meta-analysis, they were transformed using the formulas recommended by the Cochrane Handbook for Systematic Reviews of Interventions . The folThe Cochrane Collaboration’s tool for assessing risk of bias was applied independently by two authors . Informa2 test [2 value was less than 50%, then the fixed effect model (FE) was applied. The Mantel–Haenszel method was used to calculate the RR, and the inverse variance method was used to determine the WMD. The generic inverse-variance method was used to calculate the HR and rate ratio [Relative risk (RR) was used as a summary statistic for dichotomous data, and the weighted mean difference (WMD) was used for continuous data. The hazard ratio (HR) was used for time-to-event data. The rate ratio was used in instances in which it was possible for participants to experience more than one event. Statistical heterogeneity among studies was quantified using the I2 test . Intentite ratio . All sumte ratio .Subgroup analyses were performed based on the developmental statuses of the paediatric recipients  was performed with TSA Viewer Version 0.9 Beta ((c) Copyright Copenhagen Trial Unit, 2011) to control for the risk of errors for a given outcome and to help clarify whether additional trials are required. The statistical method was described in the following literatures –30. MoreRevMan 5 was not designed to analyse studies with no events in either intervention group in meta-analyses performed to determine relative risks. Exclusion of zero-event studies was unjustified and may have inflated the magnitude of the pooled treatment effects . TherefoThe article selection process is summarised in The details of the five included studies are presented in The mean difference in the ΔHSDS indicated a low risk of bias for all five studies; thus, the summary effect was also at a low risk of bias. The RR of AR was at a low risk of bias for two studies and at an unclear risk for three studies, suggesting plausible bias in the summary effect. Details on the assessment of risk of bias are provided in supporting information . All stu2 = 76%). The funnel plot was grossly symmetrical .Following stratification by developmental status, the ΔHSDS was significantly higher in the prepubertal patients undergoing the SAW regimen (WMD = 0.64) , and theWhen the subgroups were stratified by the time of initiation of steroid withdrawal, an increase in the ΔHSDS was identified in the SAW group compared with the steroid-based group in the early withdrawal subgroup . HoweverAll five studies reported the rate of AR, which was confirmed by renal biopsy (performed if indicated or according to the established protocol). Clinically defined AR was also combined with biopsy-proven AR in the meta-analysis. The rates of T-cell-mediated and antibody-mediated AR, as well as borderline changes of AR, were summed for analysis. Overall, there was no significant difference in the risk of AR between the SAW and steroid-based regimens . The funNo adequate AR data stratified by developmental status were provided in any of the five studies; thus, meta-analysis could not be performed. However, subgroup analysis of all studies indicated that there were no significant differences between the SAW and steroid-based groups within either the prepubertal or pubertal subgroup , 16,17.Subgroup analysis based on the time of initiation of steroid withdrawal revealed a significantly reduced risk of AR in the recipients with late steroid withdrawal .2, 95% CI -7.38–7.20, P = 0.98, I2 = 0%), but significant differences were observed at 1 year post-withdrawal  and 2 years post-withdrawal . The eGFR distribution in the TWIST study was likely skewed at 1 or 2 years after withdrawal [Renal allograft function, as indicated by the eGFR, which was calculated using the Schwartz formula, was reported as an outcome in 4 studies ,12,16,17The patient and graft survival data reported by the five studies were not sufficient for estimating HRs due to the lack of event distribution . In all 2 = 0%). The results for other adverse events are presented in The follow-up period for assessing adverse events ranged from 1 to 3 years. Significant reductions in the risks of NODAT and hypertension were observed in the SAW group compared with the steroid-based group . Quantitrequired information size were trustworthy. The results within either the efficacy area or futility area were also trustworthy, even though the pooled sample size did not reach the required information size. Further studies are required to confirm the reliability of the other results (labelled as not reached). Details on the evidence quality assessment and TSA are provided in supporting information , whereas the effect appeared to be lost over 1 year post-withdrawal (insufficient evidence). In addition, sufficient evidence was obtained to conclude that the SAW regimen did not increase the risk of AR or damage graft function within one to three years after steroid withdrawal. Further, the SAW regimen did not decrease graft or patient survival, and it likely reduced the risks of hypertension and NODAT and therefore the cardiovascular risk. However, the evidence was not sufficiently robust.The results of this study should be considered with caution in clinical practice due to inconsistencies among the study subjects. More than 70% of the participants were Caucasian, approximately 12% were African, and less than 5% were Asian. Variations in genetic background across races are likely to alter the outcome of the SAW protocol. Moreover, a reduced panel of reactive antibodies (PRA) was required in the recipients prior to KTx in two studies (≤20% ), whereaThe magnitude of post-transplant growth in the prepubertal recipients receiving the SAW regimen was greater than that in the pubertal recipients. This increase was attributed in part to the greater potential for growth, shorter duration of ESRD and different causes of ESRD in the prepubertal recipients. Moreover, there was less heterogeneity within the prepubertal and pubertal subgroups, whereas that among all paediatric recipients was large. A similar phenomenon was observed in subgroup analysis based on either different time points post-withdrawal or different times of initiation of steroid withdrawal. These results suggest that the heterogeneity in the post-transplant growth results can be explained partly by differences in developmental status, the time after steroid withdrawal and the time of initiation of steroid withdrawal.The current meta-analysis demonstrated no increase in the overall risk of AR, in contrast with the significant increase observed in the SAW group in a previous meta-analysis of adult renal transplant recipients  . The difImportantly, the risks of NODAT and hypertension were decreased with the SAW regimen, suggesting that future cardiovascular risk would be lowered. These results are in accordance with the adult meta-analysis, which demonstrated significant reductions in the risks of hypertension, NODAT and hypercholesterolemia with the SAW protocol . The oveThe current meta-analysis has several limitations. First, the limited number of participants in each subgroup who were stratified by factors such as the time of initiation of steroid withdrawal or developmental status did not allow for sufficient power to be achieved to identify differences between the SAW and steroid-based groups for the endpoints and could have led to substantial bias. Second, one to three years of follow-up was acceptable when evaluating the risk of AR but may have resulted in underestimation of the adverse effects of the SAW protocol (including the increased dosage of concomitant immunosuppressants or the administration of new drugs), for example, chronic rejection and CNI nephropathy. Third, the limited number of studies was not sufficient to determine publication bias.Based on the results of this meta-analysis, we suggest that the SAW regimen is justified in select paediatric renal allograft recipients because it provides significant benefits in post-transplant growth within the first year post-withdrawal with minimal effects on the risk of AR, graft function, and graft and patient survival within 3 years post-withdrawal. These select paediatric recipients should have the following characteristics:Prepubertal;Caucasian;Primary disease not related to immunological factors;De novo kidney transplant recipient;Low PRA.More trials are required to confirm the effect of the SAW regimen on post-transplant growth past 1 year post-withdrawal. Its effect on the growth of pubertal recipients should also be further evaluated.S1 Fig+/Green, low risk;? /yellow, unclear risk. Abbreviation: AR, acute rejection.(TIF)Click here for additional data file.S2 FigAbbreviations: SAW, steroid avoidance or withdrawal; SB, steroid-based; SE, standard error; MD, mean difference. Produced by RevMan Version 5.2.(TIF)Click here for additional data file.S3 FigAbbreviations: SAW, steroid avoidance or withdrawal; SB, steroid-based; SE, standard error; MD, mean difference. Produced by RevMan Version 5.2.(TIF)Click here for additional data file.S4 FigAbbreviations: SAW, steroid avoidance or withdrawal; SB, steroid-based; SE, standard error; MD, mean difference. Produced by RevMan Version 5.2.(TIF)Click here for additional data file.S5 FigAbbreviations: AR, acute rejection; SAW, steroid avoidance or withdrawal; SB, steroid-based; SE, standard error; RR, relative risk or risk ratio. Produced by RevMan Version 5.2.(TIF)Click here for additional data file.S6 FigA required information size of 708 patients was calculated based on a mean difference (MD) of the ΔHSDS of 0.4 between the SAW and SB groups; a variance of 0.9; a type I error (α) of 5%; a type II error (β) of 20%; and heterogeneity of 75%. Abbreviations: ΔHSDS, change in height z-score; SAW, steroid avoidance or withdrawal; SB, steroid-based. Produced by TSA Viewer Version 0.9 Beta.(TIF)Click here for additional data file.S7 FigA required information size of 601 patients was calculated based on an observed mean difference (MD) of the ΔHSDS of 0.22 between the SAW and SB groups; a variance of 0.75; a type I error (α) of 5%; a type II error (β) of 20%; and an observed heterogeneity of 19%. Abbreviations: ΔHSDS, change in height z-score; SAW, steroid avoidance or withdrawal; SB, steroid-based. Produced by TSA Viewer Version 0.9 Beta.(TIF)Click here for additional data file.S8 FigA required information size of 244 patients was calculated based on an observed mean difference (MD) of the ΔHSDS of 0.60 between the SAW and SB groups; a variance of 3.3; a type I error (α) of 5%; a type II error (β) of 20%; and an observed heterogeneity of 0%. Abbreviations: ΔHSDS, change in height z-score; SAW, steroid avoidance or withdrawal; SB, steroid-based. Produced by TSA Viewer Version 0.9 Beta.(TIF)Click here for additional data file.S9 Fig2 = 25%. Abbreviations: SAW, steroid avoidance or withdrawal; SB, steroid-based. Produced by TSA Viewer Version 0.9 Beta.A required information size of 880 patients was calculated based on an observed acute rejection incidence of 25% in the SB group in meta-analysis (Pc); a relative risk reduction (RRR) of -40% in the SB group; a type I error (α) of 5%; a type II error (β) of 20%; and a heterogeneity of I(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOC)Click here for additional data file.S4 File(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


This meta-analysis suggested that the polymorphisms in the iNOS gene were not associated with cancer risk.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(Ser It has been widely accepted that cancer was one of the important causes of dead all over the world, characterized by angiogenesis and inflamation3456789608 Leu) polymorphisms and polymorphic (CCTTT)n repeats.According to previous records, iNOS has played an important role in angiogenesis, stimulation of proliferation, and inhibition of apoptosisThus far, previous studies concerning association between iNOS gene polymorphism and risk of cancer are limited and rather conflicting3456789608 Leu) polymorphism and two studies5The process of the search was shown in 345678934678608 Leu) polymorphisms and polymorphic (CCTTT)n repeats and risk of cancer. In subgroup analysis for C150T (Ser608 Leu) polymorphism by ethnicity  among Asians. There was no significant publication bias according to funnel plot .608 Leu. The iNOS Ser608 Leu allele (C > T polymorphism) leads an amino acid alteration in a regulatory domain of the enzymeAs a production of nitric oxide (NO), iNOS is produced during inflammation by macrophagesin vivo or in vitro with tumor18Except detection the expression of iNOS H pylori-related gastric cancer3H 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.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 gene5This 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.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  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.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.The exclusion criteria were defined as: (1) abstracts, reviews and studies on animal; (2) useless data reported, genotype number or frequency was not included; and (3) genotype distribution in the control population not consistent with HWE.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.The review and analysis were guided to conduct by the PRISMA statement for preferred reporting of systematic reviews and meta-analysisI2 statistic interpreted as the proportion of total variation contributed by between-study variation. If there was significant heterogeneity, the random effects model would be used to analyze the pooled ORs21P value less than 0.05 for any test was considered to be statistically significant.Odds ratios (ORs) with 95% confidence intervals (CIs) for genotypes and alleles were used to assess the strength of association between iNOS gene polymorphisms and risk of cancer. The ORs were conducted for the allele contrast, codominant model, recessive model, and dominant model, respectively. Heterogeneity was examined with How to cite this article: Jiao, J. et al. Lack of association of the iNOS gene polymorphism with risk of cancer: a systematic review and Meta-Analysis. Sci. Rep. doi: 10.1038/srep09889 (2015).


Coronary artery disease (CAD) is one of the frequent cardiovascular mortality causes in the world. Common risk factors explain only about half the risk of CAD. The healthy familial predisposition to CAD, combined with advances in genetic analysis, has led to a number of studies in recent years making an effort to identify the genetic factors that influence the risk. The approach taken by most studies was to examine the association of naturally occurring genetic polymorphisms in candidate genes with risk of or severity of CAD. Endothelial nitric oxide synthase (eNOS) is important for vascular and tissue protection and is found in endothelial cells that encompass the entire vasculature, including the vessels in the heart. Nitric oxide (NO) is produced in a catabolic reaction in the endothelial cells, neurons, glia and macrophages by nitric oxide synthase (NOS) isoenzymes. eNOS is a subgroup of this family of enzymes that catalyses the production of nitric oxide (NO) from L-arginine and oxygen, which leads to vascular relaxation by activating the guanylate cyclase. This finally induces smooth muscle relaxation. The aim of this study was to investigate the allelic frequency and the genotypic distribution of the variable number of tandem repeat 27 (27 VNTR) gene polymorphism in intron 4 of the eNOS (eNOS 4a/b) gene in Thrace region, to compare CAD patients with appropriate healthy controls and to correlate the genetic findings with CAD subtypes. The study group included 281 (153 subjects with CAD and 128 controls) patients. The eNOS polymorphism was identified with a polymerase chain reaction. Genotypes were defined as aa, ab and bb according to the presence of a and b alleles. In this case–control study, we found that there was sensible correlation between eNOS gene intron 4a/b VNTR polymorphism and the risk of CAD in Thrace region of Turkey. However, there was no major difference for the genotype distribution and the allelic frequency among the CAD subtypes. Further studies on the interaction of such genes are needed to clarify the association between eNOS 4a/b polymorphism and CAD patients. Coronary artery disease (CAD) is the major cause of cardiovascular mortality worldwide. Among many traditional risk factors for CAD development, positive family history is now being considered as a significant risk factor. Atherosclerosis is the result of a defective endothelial function and an essential factor for the development of CAD. NumerousA total of 153 subjects with CAD and 128 controls were enrolled in this study. The eNOS 4a/b was genotyped using a polymerase chain reaction (PCR) method. All the CAD patients were examined by the cardiologist and the diagnosis of CAD was established in accordance with the criteria of international classification. The control groups consisted of healthy volunteers. The history of hypertension, smoking habit and the existence of cardiovascular disease in the family of the CAD patients and controls was recorded. The study was approved by the local ethic committee and informed consent was obtained from the study cases.2. All reagents for PCR amplification and gel electrophoresis were purchased from Fermentas Life Sciences .DNA was isolated from peripheral blood, collected into tubes containing ethylenediaminetetraacetic acid (EDTA) by eZNA (EaZy Nucleic Acid Isolation) blood DNA kits . eNOS 4a/b was identified using a PCR technique. eNOS intron 4a/b polymorphism was determined by PCR using oligonucleotide primers  DNA Thermal Cycler. The thermocycling procedure consisted of initial denaturation at 95 °C for 25 s, annealing at 56 °C for 35 s and extension at 72 °C for 40 s .Figure The PCR products were analysed using 2.5% agarose gel electrophoresis at 90 V for 1 h and visualized by ethidium bromide staining. The large allele eNOS 4b contains five tandem 27 bp repeats, and smaller allele eNOS 4a contains four repeats. The sizes of the PCR products were 393 and 420 bp for eNOS 4a and eNOS 4b alleles, respectively .Figure 2 test. Distribution of genotypes within groups was in accordance with the distribution predicted by the Hardy–Weinberg equilibrium model. Continuous variables among the genotypes were compared with the use of ANOVA and a post hoc Tukey test. Independent sample t-test was used to compare continuous variables between the patients and the controls. The odds ratio (OR) with a 95% confidence interval (CI) of the possible risk factor for CAD was evaluated by binary logistic regression analysis. Categorical data were compared by χ2 and Fisher's exact test where appropriate. The triglyceride pressure measurements significantly deviated from a normal distribution by Kolmogorov–Smirnov test. Therefore, log-transformed values were used for these parameters. Univariate and logistic regression analyses were used to evaluate the significant associations between the study parameters and the genotypes. All tests were two sided and a P value < 0.05 was considered as significant.All statistical analyses were performed with the SPSS 15.0 software. Data are presented as mean ± SD, median or per cent frequency. The frequencies of the genotype distribution  between the patients and the controls were compared by χP = 0.002); HDL (P < 0.001), LDL (P < 0.001), TG (P = 0.003) and TC (P < 0.001); and the frequencies of hypertension (P = 0.043); current smoker (P = 0.01) and family history of CAD (P = 0.029) between the CAD and control groups. The genotype frequencies were in agreement with those predicted by the Hardy–Weinberg equilibrium.Our study included 281 subjects: 153 CAD patients and 128 controls . All parP < 0.001) after adjustment of several potential confound factors.P = 0.404). Patients carrying ab heterozygote genotype have a 2376-fold CAD risk .Control and CAD groups’ genotypes and the risk of developing CAD are shown in P < 0.05).eNOS gene polymorphism may influence the functional activity of the enzyme and has modulating effects on atherogenesis. Previous studies have indicated phenotypic significance of eNOS 4a/b polymorphism in various conditions and its indirect effects on atherogenesis. In additP < 0.05). Fatini et al. [n = 30). This observation has been affirmed by Ichihara et al.[n = 104). Hence, the harmful effects of eNOS 4a allele on atherosclerosis may appear independently from conventional risk factors. Asakimori et al. [eNOS gene polymorphism may have an influence on the functional activity of the enzyme, and, moreover, it has modulating effects on atherogenesis. Phenotypic significance of eNOS 4a/b polymorphism in various conditions and indirect effects on atherogenesis were shown in previous studies. Furthermi et al.  showed tra et al. In Ichihi et al.  reportedi et al.  could noEstablished cardiovascular risk factors including dyslipidaemia, smoking, hypertension and diabetes mellitus have been incorporated into algorithms for risk assessment in the general population, but thesIn the present study, we observed that eNOS 4a/b expression was increased in the CAD group compared to the control group. The results also exhibited that eNOS intron 4a/b 27 VNTR polymorphism was associated with CAD, and homozygote 4a4a genotype frequency was significantly higher in the CAD group compared with the control group. Furthermore, the results stated that heterozygote 4a4b genotype carriers have increased the level of eNOS expression compared with the same genotype in the control group which might have contribution to reduce the risk of CAD.In this case–control study, we found that there was a reasonable relation between eNOS gene intron 4a/b VNTR polymorphism and the risk of CAD. We also exhibited that eNOS intron 4b/4a 27 bp VNTR polymorphism was associated with CAD, and aa genotype frequency was significantly higher in CAD people in comparison with normal people. The limitation of our study was especially selected population, so that it does not represent common population but patients referred for CAD. This finding is potentially important; however, more detailed analyses are necessary to elucidate the associations of genetic abnormalities and CAD and its risk factors.


To summarize the relationship between type 2 diabetes mellitus (T2DM) and risk of colorectal adenomas (CRA), we performed a meta-analysis of observational studies.To find studies, we searched PubMed, Embase, the Cochrane Library, Web of Science and conference abstracts and related publications for American Society of Clinical Oncology and the European Society of Medical Oncology. Studies that reported relative risks (RRs) or odds ratios (ORs) with 95 % confidence intervals (CIs) for the association between T2DM and risk of CRA were included. The meta-analysis assessed the relationships between T2DM and risk of CRA. Sensitivity analyses were performed in two ways: (1) by omitting each study iteratively and (2) by keeping high-quality studies only. Publication bias was detected by Egger’s and Begg’s tests and corrected using the trim and fill method.This meta-analysis included 17 studies with 28,999 participants and 6798 CRA cases. We found that T2DM was a risk factor for CRA , and also for the advanced adenoma . Patients with existing T2DM  or newly diagnosed T2DM  have a risk of CRA. Similar significant results were found in retrospective studies  and population based cross-sectional studies , but not in prospective studies .Our results suggested that T2DM plays a risk role in the risk of developing CRA. Consequently, medical workers should increase the rate of CRA screening for T2DM patients so that they can benefit from behavioural interventions that can help prevent the development of colorectal cancer. Additional, large prospective cohort studies are needed to make a more convincing case for these associations.The online version of this article (doi:10.1186/s12885-016-2685-3) contains supplementary material, which is available to authorized users. Diabetes mellitus (DM) is the fourth or fifth leading cause of death in developed countries and one of the biggest threats to human health worldwide . More thResearch on risk factors for CRA has focused on several epidemiological factors, including smoking , alcoholTwo investigators (FY and YG) independently conducted a systematic literature searches on January 10, 2016 in PubMed, Embase, the Cochrane Library and Web of Science without limiting the publication date range. The following search terms were used: (diabetes mellitus OR diabetes OR diabetic OR glucose) AND  AND (adenomas OR adenoma OR adenomatous OR polyp). No language restrictions and any other limitations were imposed. Conference abstracts on the websites of American Society of Clinical Oncology’s (ASCO) and the European Society for Medical Oncology’s (ESMO) annual meetings were also searched, along with the reference lists of the identified publications. Additional file The titles and abstracts of all of the studies from the searches were screened independently by three reviewers . To be included in this meta-analysis, studies had to be at least one of the following criteria: (1) retrospective or perspective observational study of the association between diabetes mellitus and CRA, or (2) a study reporting the relative risks (RRs) or odds ratios (ORs) for T2DM on CRA with 95 % confidence intervals (95 % CIs) adjusted for gender, age, or other factors. Studies reporting on the CRA recurrence were excluded.Data extraction was performed by three reviewers , and verified independently for accuracy by a forth reviewer (JH). The following information was collected for each study: title and author, publication year, population, location, sample size, proportion of males and covariates controlled for by matching or multivariate analysis. For studies that reported several multivariate adjusted ORs, the effect estimate that adjusted for the maximum potential confounders was selected. Two investigators (FY and ZJ) conducted a quality assessment using the 9-star Newcastle-Ottawa Scale (NOS) , which w2) statistic [p < 0.10 [We examined the relationship between T2DM and CRA risk on the basis of the adjusted RRs and ORs and corresponding 95 % CI published in each study. A fixed effects model was used to estimate the pooled RR and OR with 95 % CIs if there was no evidence of heterogeneity; otherwise, a random effect model was used , 24. Bectatistic . Statistp < 0.10 .Several methods were used to test and adjust for potential publication bias. Visual inspection of funnel plots was performed, and the Egger’s regression test  and Beggp < 0.05 Stata  was used for all analyses.All statistical tests were two-sided and regarded as statistically significant at Until January 10, 2016, 2522 records were retrieved by using our search strategy. After reviewing the titles and abstracts, 113 articles were further evaluated by reviewing the full texts. Of those remaining articles, we excluded studies that : (1) reported the data of adenoma recurrence were excluded , 32, (2)I2 = 65.6 %, P < 0.001). Figure The summary RR of diabetes on CRA was statistically significant . Evidence of the heterogeneity was identified  and Egger’s regression test (p = 0.003) results showed potential publication bias that is described in Fig. The Begg’s rank correlation test  might take effect in the adenoma–carcinoma process. High insulin levels could promote tumor growth , 45, 46.Some studies reported a difference in risk between males and females , 53–55; Our analysis revealed that with T2DM have about a 5 % higher risk of CRA than newly diagnosed diabetes patients, revealing the duration of T2DM as a risk factor for CRA. A possible explanation is that known T2DM patients’ bowels are exposed to hyperinsulinemia or a hyperglycemic environment for a longer time, and such hormonal or metabolic abnormalities (according to former study ) could aSeveral limitations of in this meta-analysis that should be taken into consideration. First, results for several subgroups, such as gender and adenoma sub-site subgroup, were based on a limited number of studies. Therefore, we cannot rule out the possibility that insufficient statistical power is present. Second, in the present analysis, some small studies with inverse associations between T2DM and risk of CRA risk seemed to be suppressed. The presence of possible publication bias could have led to an overestimation of the effect of T2DM on CRA risk. However, the adjusted result was comparable after trim and fill method corrections. Third, we could not account for all of the confounding factors in the meta-analysis, though most confounders were adjusted in the original RRs. Many factors might induce the adenomas, such as age, ethnicity, inactivity, regular aspirin use, obesity, and family history of CRA, and menopausal status. We could not control for these covariates because of lack of relevant data. Relevant studies with additional data on these other factors may be found by searching by searching beyond the sources used for this study. Furthermore, we could not determine whether using insulin as a therapy for T2DM is an important factor because CRA risk might be altered by hyperinsulinemia, thought to be an important promoter of carcinogenesis , 63. At In conclusion, the results of our meta-analysis indicated that patients with T2DM have higher risks for the development of CRA, which is an important inducement for colorectal cancer. Our study has important implications for clinical and public health. Because T2DM and CRA are prevalent in the developed and developing countries , medical


STK39) is one of the promising hypertension-susceptibility genes identified by a genome-wide association study in 2009, whereas subsequent validation in other ethnic groups is unsatisfactory, with inconsistent and inconclusive findings. We therefore aimed to meta-analytically assess the risk prediction of STK39 three polymorphisms, rs6749447, rs35929607 and rs3754777, for primary hypertension. Literature search and data collection were independently completed by two authors. Nine articles were pooled in this study. Overall analyses failed to see any significant associations of rs6749447, rs35929607 and rs3754777 with hypertension risk , and there was evident heterogeneity for three comparisons (I2 > 80%). Meta-regression analyses indicated that smoking was a significant risk factor for the association of rs3754777 with hypertension (P = 0.017). As reflected by the Begg’s and Filled funnel plots, as well as Egger’s tests, there were low probabilities of publication bias. In conclusion, our meta-analytical findings suggest that STK39 might not be a hypertension-susceptibility gene.Serine/threonine kinase 39 gene ( STK39) was first identified as a hypertension-susceptibility gene by a genome-wide association study in 2009STK39 can predispose individuals at risk for elevated blood pressure23STK39 gene encodes a serine/threnine kinase termed as a Ste20-related proline/alanine-rich kinase (SPAK). Compelling evidence from in vitro studies indicates that SPAK binds to and phosphorylates Na+-dependent cation chloride cotransporters, which can mediate renal NaCl reabsorption and further control salt transport in osmotic cell volume regulation56STK39 is located on chromosome 2q24.3, comprises 18 exons and spans approximate 300 kbSTK39 (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=27347). Thereof, three intronic SNPs, rs6749447, rs35929607 and rs3754777, have been extensively evaluated in association with hypertension, yet with inconsistent and inconclusive findings, likely due to the genetic heterogeneity of populations selected, the possible small effect of single SNP on hypertensive risk, the relatively small sample size in individual studies, and the lack of evidence in populations other than Asian and White origins39STK39, according to the guidelines listed in the PRISMA  statement .Since serine/threonine kinase 39 gene . In addition, considering the high misclassification rate of restriction fragment length polymorphism (RFLP) method used by two studies12As discrete genotype counts are not usually available in some studies, only alleles were calculated and pooled in this meta-analysis . OverallEgger = 0.955), rs35929607 (PEgger = 0.187), and rs3754777 (PEgger = 0.986), and there were no missing studies as indicated by the Filled funnel plots in As shown in the Begg’s and Filled funnel plots , as wellSTK39, for primary hypertension, and none of these SNPs contributed to the significant risk of hypertension. Even though, it is of interest to note that rs3754777 might interact with smoking to make individuals more susceptible to experience hypertension. To our knowledge, this is so far the most comprehensive meta-analysis pivoting STK39 genetic alterations and hypertension risk from medical literature.The aim of this meta-analysis was to assess the risk prediction of three promising SNPs, rs6749447, rs35929607 and rs3754777 in et al. examined only rs3754777 in STK39 along with its strongly-linked proxies (rs2063958 and rs35929607), and found that rs3754777 was a significant risk factor for primary hypertension in populations of Caucasian and East Asian ancestrieset al.http://www.broadinstitute.org/mammals/haploreg/haploreg.php), rs2390639 is a more suitable candidate to surrogate rs3754777 as their linkage disequilibrium coefficient D’ reached 0.99. However, the two SNPs, rs2063958 and rs2390639 in the study by Kidambi et al.et al.STK39 to hypertension, and no observable significance was noticed, suggesting that multiple SNPs in STK39 mediate blood pressure regulation, with each exerting small effects under a certain environmental condition.A previous meta-analysis by Xi STK39 gene, and further control the salt transport.It is worth noting in this study our meta-regression analyses suggested a possible interaction between rs3754777 and smoking in predisposition to hypertension. There is a wide recognition that smoking is an established risk factor for hypertension18Several drawbacks for the present meta-analysis need to be acknowledged. First, the sample size in this study is not large enough to ensure adequate statistical power for obtaining a convincing estimate. Second, only hypertension as a binary trait was analyzed and blood pressure as a continuous trait was not due to the insufficient data across qualified studies. Third, the conclusion of this study was merely based on case-controls studies, which might hinder the perhaps cause-effect relationship. Fourth, in spite of no indication of publication bias, there was strong evidence of between-study heterogeneity, and interpreting heterogeneity is still a challenge, as with a majority of meta-analysesSTK39 might not be a hypertension-susceptibility gene. Future studies should incorporate various lifestyle and environmental risk factors such as smoking and drinking in genetic estimation models to precisely define the role of specific genetic variants.Taken together, we through a comprehensive meta-analysis concluded that www.wanfangdata.com.cn/, a Chinese database since 1966) and CNKI  were looked through to find studies that were aimed to explore the associations of STK39 genetic variants with hypertension in human beings on October 25, 2015. The language of qualified articles was confined to English or Chinese. This search process was independently run by two authors  on the basis of predefined key terms. Search results were combined with the removal of any duplicate articles.Four databases including Medline (PubMed since 1966), Embase (Excerpta Medica Database since 1980), Wanfang  primary hypertension as the clinical endpoint; (b) case-control study design; (c) one or more SNPs of rs6749447, rs35929607 and rs3754777 under investigation; (d) detailed genotype or allele frequencies for examined SNPs between cases and controls. The selection process was completed by two independent authors , and any disagreement was resolved through discussion.From each qualified article, relevant data were abstracted by Hualing Yang and subsequently checked by Lingyang Ye, and all data were entered into a predetermined Excel spreadsheet, including the first author’s surname, year of publication, race of study subjects, study design, control source, genotyping method, a list of SNPs under study, genotype or allele counts, sample size, age, male proportion, body mass index, smoking, drinking, fasting glucose, triglyceride, total cholesterol, and high-density lipoprotein cholesterol.STK39 three SNPs for hypertension risk were pooled in a random-effects model, and odds ratio (OR) and its 95% confidence interval (95% CI) were calculated by the DerSimonian and Laird methodI2 value in terms of percentage, and significant heterogeneity was reported if the I2 value is greater than 50%, with higher values indicating stronger heterogeneityIndividual effect-size estimates of How to cite this article: Yang, H. et al. A meta-analytical assessment of STK39 three well-defined polymorphisms in susceptibility to hypertension. Sci. Rep.6, 25290; doi: 10.1038/srep25290 (2016).


Published data showed that the susceptibility of autoimmune diseases (ADs) was associated with the polymorphism rs2910164 in microRNA-146a (miR-146a). However, the results remain controversial so far. Two meta-analyses published in 2013 and 2014 came to opposite conclusions. In order to derive a more precise estimation of the relationship, we performed this meta-analysis.We searched the PubMed, OvidSP and CNKI databases  and extracted data from eligible studies. The procedure of meta-analysis was performed by using the Stata 12.0 software. Random effect model or fixed effect model were chosen respectively, according to the between study heterogeneities.p = 0.024) and dominant model , respectively. Meanwhile, in other disease subgroup, significant increased risks of C allele, CC genotype and GC+CC genotype were found in allele model , homozygote model  and dominant model , respectively.A total of 24 case-control studies, 11 more than previous meta-analysis on this topic, were involved. We took stratified analyses by different ethnicities and different types of diseases in different genetic models. In Caucasian subgroup, significant increased risks of GC genotype and GC+CC genotype with ADs susceptibility were found in heterozygote model (GC vs GG, OR = 1.38, 95% CI 1.04–1.83, MiR-146a rs2910164 G>C polymorphism was associated with the susceptibility of ADs. Autoimmune diseases (ADs) are initiated by abnormal immune response to self-antigen, and then come the results including immune-mediated tissue destruction and chronic disabilities . There aMicroRNAs (miRNAs) are non-coding single-stranded RNA molecules. By binding to 3’ un-translated regions (UTRs) of targeted messenger RNAs, the miRNAs can repress, degrade or silence the gene expression , 6. TherSingle nucleotide polymorphisms (SNPs) or mutations in miRNAs may alter the expression level of the gene and the susceptibility of some diseases –13. Manyth, 2014. The searching strategy was as follow: (autoimmune OR autoimmune disease OR autoimmunity) AND (polymorphism OR polymorphisms OR variation OR variations OR mutation OR mutations OR variant OR variants) AND (Has-mir-146a OR miR146a OR microRNA-146a OR miR-146 OR miR-146a OR rs2910164). The references in the studies were also read to find additional publications on the topic. Articles included should meet the criteria below: (1) case-control study; (2) evaluation of miR-146a rs2910164 G>C polymorphism and risk of ADs; (3) available and usable data of genotype frequency. The articles were excluded if they meet the exclusion criteria below: (1) a case report, review or descriptive study; (2) a lack of normal population as controls; (3) not show the evaluation of miR-146a rs2910164; (4) duplicate data in the studies.A systematic search was performed in PubMed, OvidSP and Chinese National Knowledge Infrastructure (CNKI) databases covering all papers published prior to September 8Two authors (CL and WF) independently extracted the data from eligible studies. Different data extracted by CL and WF were checked by the third author YZ. The remained disagreements were discussed and judged by these three authors. The following information was extracted: the first author, publication year, diseases, country, ethnicity, genotyping methods, number of cases and controls, the gender distribution of cases and controls, number of genotypes and alleles, Hardy-Weinberg equilibrium (HWE) in control subjects, frequency of G allele in controls. Ethnicities were categorized as Caucasian, East Asian, Latin-American and Mediterranean. Study qualities were judged according to the criteria modified from previous publications –40 (See 2) tests. If the heterogeneity was considered significant (p<0.05), the random effect model was used to estimate the pooled OR. Otherwise, the fixed effect model was conducted. Also, logistic meta-regression analysis was carried out, if there was obvious significant heterogeneity, to explore potential sources of heterogeneity. The examined characteristics include: publication years, countries, genotyping methods, number of alleles and genotypes, number of female and male in cases, and the frequency of G allele in controls. The HWE was examined using Chi-square test with significance set at p<0.05. Sensitivity analysis was performed to evaluate the effect of each study on the combined ORs by deleting each study in each time, and to evaluate the effect of studies with low quality or without HWE on the pooled ORs by deleting these studies. Potential publication bias was determined by using Funnel plots and Begg’s test. An asymmetric plot and the p value less than 0.05 was recognized as significance. All statistical analyses were performed by Stata 12.0 software .Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated as a measure of the association between miR-146a rs2910164 G>C polymorphism and ADs risk. The widely reported genetic models, allele model, heterozygote model, homozygote model, dominant model and recessive model, were used. In addition to comparing the pooled effects among all subjects, the stratified comparisons were also conducted according to different ethnicities and different diseases. In order to evaluate the accumulation of evidence, the cumulative meta-analysis was performed. The between study heterogeneity was measured by Cochran’s (Q) and Higgins’s , but the p value of HWE were not less than 0.01. And there was not enough data in another study to generate the HWE in control. The detail characteristics are shown in Within the 21 articles, five kinds of genotyping methods were used. There were 4 racial included, Caucasian, East Asian, Latin-American and Mediterranean. According the different types of diseases, these studies were divided into five subgroups. The studies on Rheumatoid Arthritis (RA), Psoriatic Arthritis (PsA), Juvenile RA (JRA) and Juvenile Idiopathic Arthritis-Enthesitis-Related Arthritis (JIA-ERA) were included into Inflammatory Arthritis (IA) subgroup. The studies on Ulcerative Colitis (UC) and Crohn’s Disease (CD) were included into Inflammatory Bowel Disease (IBD) subgroup. Among these studies, the patients with Behcet’s Disease (BD), Vogt-Koyanagi-Harada syndrome (VKH), Fuchs Uveitis Syndrome (FUS) and Pediatric Uveitis (PU) were all suffering uveitis, which was a common syndrome of ADs. So the Uveitis subgroup was formed. The remained studies, except that for Systemic Lupus Erythematosus (SLE), were included in other diseases subgroup. There were 5 studies not in HWE in control groups (p = 0.024) and dominant model , respectively , homozygote model  and dominant model , respectively (p = 0.057) in IBD subgroup . The meta-regression analysis was conducted to further explore sources of heterogeneity. We assessed allele comparison by potential sources of publication year, country, genotyping methods, number of genotypes and alleles, and number of female and male in cases. None of the potential sources above could explain the heterogeneity by meta-regression analysis. However, when we compared the frequencies of G allele in controls, we found that the heterogeneity could partly (Adjusted R2 = 32.7%) explained by the variation of frequencies of G allele in controls.The heterogeneities among studies were obvious in the overall comparisons (Ip = 0.261) and Begg’s test (p = 0.862)  .Solid evidences had shown that miRNAs played important roles in the regulation of cell differentiation, proliferation, metabolism, apoptosis and tumorigenesis . MiR-146One SNP of miR-146a, rs2910164 G>C, was found not only related to cancer, but also related to some kinds of ADs . While tIn this meta-analysis, we enrolled 24 studies and pooled the corresponding data including 7591 cases and 9677 controls, and all data of these samples were available in the original publications. Our analysis revealed that there was not any significance of pooled OR. However, in stratified analyses, we found GC genotype and GC+CC genotype were significantly related to the increased susceptibility of ADs in Caucasian subgroup. As in other diseases subgroup, significantly increased risk was observed to be associated with C allele, CC genotype and GC+CC genotype in allele model, homozygote model and dominant model, respectively. All these differences were not found in the two previous meta-analyses , 36. TheIn cumulative meta-analysis, we found the significance of ORs after new case-control studies published in 2013 and 2014 enrolled. These data demonstrated the correlation of rs2910164 and susceptibility of ADs. In addition, not only sensitivity analysis by iteratively removing one study at a time, but also analysis removing three studies , 24, 34 Taken together, our data demonstrated that the miR-146a rs2910164 G>C polymorphism was related to the susceptibility of ADs and our findings should be validated by future well-designed large studies.S1 PRISMA Checklist(DOC)Click here for additional data file.S2 Checklist(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Helicobacter pylori associated inflammatory response, which may contribute to individual differences in the outcome and severity of the disease. Interleukin4 is a typical pleiotropic T helper 2 (Th2) cytokine and is a critical mediator of Th1/Th2 balance. It is involved in the regulation of inflammation-mediated carcinogenesis in human organs, including gastric cancer.Gastric cancer (GC) is the fifth most common malignancy and remains a considerable public health burden worldwide. Genetic variations in genes encoding cytokines and their receptors influence the intensity of the The present retrospective case control study was undertaken to evaluate the association of IL4 intron 3 VNTR polymorphism with the susceptibility to GC in a south Indian population from Telangana state.A total of 182 patients with diagnosed GC and 326 randomly selected healthy controls were enrolled in the present study. Genomic DNA was extracted from peripheral leukocytes and genotyping was determined by PCR-based assay. Association between genotypes and gastric cancer was examined by unconditional logistic regression analysis.vs. 3R/3R: adjusted odds ratio (AOR) = 1.90, 95% confidence interval (CI) = 1.23–2.95 P = 0.004 and 2R/2R vs. 3R/3R: AOR (95%CI) = 2.96 (1.29–6.82), P = 0.011]. Furthermore, a significant increased risk of GC was found for the 2R allele carriers (3R/2R + 2R/2R) compared with the 3R/3R genotype (AOR (95%CI) = 2.04 (1.35–3.10), P = <0.000). The IL4 2R allele frequency was 0.28 among the GC group and 0.18 among the controls, and the difference was statistically significant (P = <0.000).The variant 3R/2R and 2R/2R genotypes of IL4 exon3 VNTR polymorphism had about 1.9 fold and 3fold increased GC risk, respectively, when compared with 3R/3R genotype [3R/2R The present study revealed an association of 2R allele and 2R carrier genotypes in the etiopathogenesis of GC in south Indian population. Despite declining incidences and mortality rates observed worldwide, gastric cancer (GC) is the fifth most common cancer  and ranks third as the cause of cancer related mortality , . Its patHelicobacter pylori (HP) infection, exogenous environmental and endogenous genetic factors . Moderate and chronic smokers were categorized by using the median pack-year value (25 Pack years) as the cut points. Information collected on the frequency of alcohol use per week and the total duration (in years) of alcohol consumption was used to derive drinking frequency-years [(times of drinking-per week)*(years drinking)]. Alcoholics were divided into moderate and chronic groups by using the median frequency-years as the cut points. Deviations from the Hardy–Weinberg equilibrium (HWE) were tested using the xon 3.1.2  and R paon 3.1.2 .P<0.000). When divided into moderate and chronic smokers there was 2- and 3.7- fold increased risk of GC, respectively with reference to nonsmokers. Tobacco chewers were at about 4 fold increased risk of GC when compared with nonchewers. Similarly with respect to alcohol consumption, we found significant difference with overall alcoholic group (~2.2fold) as well as with moderate (1.9 fold) and chronic (2.9 fold) alcoholic groups when compared to nonalcoholic group.Epidemiological characteristics of patients and controls are presented in 0.004) and 2.96 fold  risk of having GC, when compared with the 3R/3R i.e. major genotype. Likewise, there was significant increase in frequency of 2R alleles  in GC patients group in comparison to the control group. When we combined 3R/2R and 2R/2R genotypes, assuming a dominant allele effect, the combined (3R/2R +2R/2R) variant genotypes were associated with 2.04 fold  increased risk of GC for 2R allele carriers. Based on the values of Akaike information criterion (AIC), dominant model with least AIC value was found to be best genotypic model indicating about 2 fold increased risk for 2R allele carriers.The genotypic and allelic frequencies of the IL-4 intron 3 VNTR polymorphism in patients with GC and controls are shown in We examined whether the associations of this polymorphism with gastric cancer risk were modified by interaction with other risk factors including age, gender, smoking, tobacco chewing and alcohol consumption. To maximize power in the cross-classification/interaction analysis, subjects with heterozygote (3R/2R) and homozygous (2R/2R) variant genotypes were combined and ORs were expressed with reference to the non variant genotype (3R/3R) and the first category of the covariates. Cross-classification analysis of IL4 VNTR polymorphism with demographic variables is presented in The association between polymorphism and gastric cancer stratified on demographic features like age, sex, tobacco chewing, smoking and alcohol use are presented in In the present study, we investigated the association between IL4 VNTR gene polymorphism and risk of gastric cancer in a South Indian population. The most important result of our study was the significant association of 2R allele and 2R carrier genotypes (i.e. 3R/2R or 2R/2R) with increased risk of GC, indicating the possible association of IL4 VNTR polymorphism with GC. To the best of our knowledge, this is the first association study that attempted to evaluate the association of IL-4 gene VNTR polymorphism on GC in a south Indian population.H. pylori-induced gastric mucosal inflammation and atrophy by decreasing interferon γ (IFN-γ) and other Th1-type cytokines [H. pylori infection. Th2 T-cell response, represented by IL4, plays a protective role in the development of gastric cancer. Further IL-4-deficient mice infected with H. pylori show severe gastric inflammation compared with wild-type mice [via its suppression of inflammation and angiogenesis and directly inhibits the growth of human melanoma, renal cell carcinoma and gastric cancer cells [GC is a classical example of inflammation induced malignancy. IL4 is an important anti inflammatory cytokine which inhibits ytokines . IL4 indype mice . In cancype mice . IL-4 iser cells . IL4 haser cells  and to ier cells . IL-4 maer cells . Thus itP = 0.018) with bladder cancer and tumour invasiveness [Several epidemiological studies have examined the association between the IL4 VNTR polymorphism and the risk of cancer, including prostate, urothelial, breast, colorectal, and gastric cancers but the results are inconsistent. In a study by Yang et al (2014) IL-4 intron 3 VNTR polymorphism was found to be associated  with early stage oral and pharyngeal carcinoma risk (OPSCC), which also interacted with alcohol consumption p = 0.024) . 2R carr . 2R carsiveness . Shekarisiveness . There wsiveness . No assosiveness . No signsiveness . Similarsiveness . In contA subject’s epidemiological/life style related factors may also play an important role in the etiology of GC and these factors may act in synergy with genetic polymorphism of IL4. As expected, addictions, such as tobacco chewing, smoking and alcohol use, were correlated with significantly increased susceptibility to GC. Further, the variant 2R carrier genotypes were found to exhibit more pronounced risk with respect to female sex, middle (45–60 years) age group, chronic smoking, tobacco chewing and chronic alcoholism indicating modulation of risk by these epidemiological factors. However, the numbers within each sub-group were too low and larger studies are warranted to exhibit significant interaction.Our study has certain limitations which need to be considered in interpreting our results. Community based Control subjects were not matched for age and gender. Although MLR was applied, effect of residual confounding can not be completely ruled out. Our sample size was relatively small which may influence the statistical power in the study, particularly for interaction and stratified analysis. We did not have detailed Clinico-pathological information of all the patients which does not permit us to analyze our data in the context of histological subtypes, grades and stages of gastric cancer. Further in the light of the large repertoire of SNPs in pro- and anti-inflammatory cytokine genes with complex interactions in a multifactorial complex disease like gastric cancer, the results of this study may be insufficient to clarify the complex interplays among a number of different genes in GC but may represent a hypothesis generating data for further investigations.In conclusion our results suggest that variant genotypes and allele of IL 4 VNTR polymorphism may significantly increase the susceptibility to GC and implicates the 2R allele as one of the potential genetic markers in the etiology of the disease in our population. However, a large confirmatory study involving other populations is warranted to understand the population-specificity and the relative contribution of this polymorphism in the etiology of gastric cancer.


XPC) gene for their correlation with urinary bladder cancer. Ala499Val and Lys939Gln polymorphisms were genotyped in 234 urinary bladder cancer cases and 258 control samples. A significant association between Ala499Val polymorphism and bladder cancer was observed ; however, Lys939Gln was unrelated . Further analysis revealed that Ala499Val was a significant risk factor only in the presence of smoking  or tobacco chewing . To further appraise the association, we undertook meta-analyses on seven studies (2893 cases and 3056 controls) on Ala499Val polymorphism and eleven studies (5064 cases and 5208 controls) on Lys939Gln polymorphism. Meta-analyses corroborated the above results, showing strong association of Ala499Val  but not that of Lys939Gln  with urinary bladder cancer risk. In conclusion, XPC Ala499Val substitution increases urinary bladder cancer risk, but Lys939Gln appears to be neutral.Compromised activity of the DNA repair enzymes may raise the risk of a number of cancers. We analyzed polymorphisms in the Xeroderma Pigmentosum, Complementation Group C ( Urinary bladder cancer (UBC) is the fifth most common malignancy in the economically advanced countriesXPC) gene encodes a 940 amino acid protein that plays an important role in the initiation of DNA repairXPC gene may alter the NER capacity and affect genetic predisposition to cancer. Many studies have shown that polymorphisms in the XPC gene are associated with the risk of esophageal squamous cell carcinoma, gastric cardiac adenocarcinoma, squamous cell carcinoma of the head and neck, breast cancer, renal cell carcinoma, urinary bladder cancer, advanced colorectal adenoma, oral squamous cell carcinoma, lung cancer, and pancreatic adenocarcinoma8Xeroderma Pigmentosum, Complementation Group C , p = 0.004] and tobacco-chewers [CT + TT ]. The association was also seen in the co-dominant models, further supporting that substitution at this locus increased the risk of UBC and that the inheritance followed a dominant model. In the case of non-smokers  and tobacco non-chewers , no association with the UBC risk was seen. The above results suggested that Ala499Val substitution was a UBC risk factor only in the presence of smoking or tobacco-chewing habits.No major difference was seen in the distribution of alternate genotypes at this locus between cases and controls . WhereasAla499Val and Lys939Gln polymorphisms were not in significant linkage disequilibrium with each other . Four haplotypes with a frequency of more than 1% were detected . The distribution of all the haplotypes, except AC, was significantly different between case and controls . AT and et al.et al.et al.et al.et al.The literature search retrieved a total of 34 relevant articles, which were subjected to systematic screening for inclusion in meta-analysis . We inclThe genotype data were heterogeneous across all genetic models; hence, the random-effects model was used to perform pooled analysis. Meta-analysis suggested that Ala499Val correlated with UBC risk in the recessive  and co-dominant models  , Table 6Looking at the striking differences in the association status among ethnically different populations, we undertook a category-wise analysis on four ethnic groups; Chinese, Indians, Americans, and Europeans using dominant, homozygous, recessive and allelic contrasts. Stratified analysis showed a significant association of Ala499Val substitution with UBC risk only in Europeans across dominant , recessive  and the co-dominant models  ; howeverThe results of TSA were consistent with those of the conventional meta-analysis. TSA using co-dominant (CC vs TT and CT vs TT) and recessive models showed that the blue line of cumulative Z-score crossed the red line sloping inwards (significance line of TSA), suggesting a significant association of Ala499Val substitution with the UBC risk . FurtherIn the case of Lys939Gln polymorphism, the results of TSA were similar to those of the conventional meta-analysis, suggesting that Lys939Gln polymorphism is not significantly associated with the UBC risk. In none of the models , the blue line of cumulative Z-score crossed the significance line of TSA. Further, TSA revealed that meta-analysis had enough number of studies (required sample size = 2974) in the pool to achieve 80% study power as it crossed the O’Brien-Fleming boundary.XPC gene for their correlation with the UBC risk. Single locus analysis revealed a strong association of Ala499Val substitution with UBC risk, supporting the previous findings on the various ethnic populations613171858111314151846In this study, we analyzed two polymorphisms in the XPC genotypes were at an increased risk of UBC when compared to non-smokers418214et al.It is known that smoking and tobacco chewing increase the risk of cancer in general. A few previous studies suggested that the smokers harboring mutated et al.et al.et al.Meta-analyses till date have analyzed XPC polymorphisms for correlation with overall cancer risk and the risk of specific cancers. Francisco et al.et al.et al.et al.)Among meta-analyses conducted specifically on UBC, Stern In conclusion, we observed a significant correlation of Ala499Val with UBC risk in Indian population, but Lys9393Gln was neutral. Meta-analysis and TSA corroborated these results, suggesting that Ala499Val was a risk factor for UBC and Lys939Gln was a neutral polymorphism. There is now sufficient evidence to conclude that XPC polymorphisms increase the risk of cancer, particularly UBC. From the literature review and a number of meta-analyses presented above, it can be postulated that Ala499Val is a more prominent risk factor for UBC and other cancers across a large number of populations; however, Lys939Gln may be largely neutral. Further analysis on the various ethnicities would validate these findings. We must admit some limitations of this meta-analysis. First, in the sub-group analysis, not all the ethnic populations were equally represented. Some of the sub-groups had higher numbers of studies in comparison to the others, which may bias the conclusion regarding the correlation status in each ethnic group. In stratified analysis, we could compare the risk factor  only in Europeans and Americans due to a lack of studies on Indians and Chinese. Second, we found Ala499Val to be a significant risk factor only in the presence of smoking or tobacco chewing; therefore, it would have been ideal to adjust pooled OR value with respect to smoking and tobacco chewing. However, due to the lack of original data for each study, we could not adjust the pooled OR with respect to age, sex and environment.A total of 234 patients with histopathologically confirmed transitional cell carcinoma of bladder and 258 healthy control subjects were recruited from the Department of Urology at the King George’s Medical University (KGMU), Lucknow. Written informed consent was obtained from each participant for personal interviews and collection of blood samples for research purpose. Age, gender, and smoking status were registered for all the patients and controls. The Institutional Ethics Committee of the KGMU, Lucknow, approved the study (ref no. XLIX ECM A-/P14). The experiments were carried in accordance with the guidelines approved for research on human samples.XPC polymorphisms were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct DNA sequencing methods. PCR was carried out in a reaction volume of 10 μl each in thin walled tubes, consisting of 1.0 μl of PCR buffer (10x)(New England Biolabs), 1.0 μl of 10 mM dNTPs (Genei), 2.0 pM of each of the forward and reverse primers, 1.0 unit of Taq DNA polymerase enzyme (New England Biolabs), and 40 ng of genomic DNA. PCR cycling was carried out in ABI Veriti thermal cycler . The primers used for K939Q polymorphism were; forward (5′ACCTGTCCAGAGTGAGGCAG3′) and reverse (5′TCAAAGGGTGAGTGGGCTTT3′) primers, and for Ala499Val were; forward-(5′TGGCCTCCAGGGTGTCTTAT3′) and reverse (5′ACTGTCAATGCCCACCACAT3′). PCR amplification conditions included denaturation at 95 °C for 5 minutes, followed by 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 67 °C for 30 seconds, polymerization at 72 °C for 40 seconds, and a final stage of polymerization at 72 °C for 7 minutes.Genomic DNA was extracted from the peripheral blood samples using a phenol-chloroform precipitation based method. The PvuII and AciI restriction enzymes (New England Biolabs) for Lys939Gln and Ala499Val, respectively. After digestion, the samples were run on a 3% agarose gel and samples were classified as homozygous for Lys type (493 bp fragment), homozygous for the Gln type alleles (223 and 270 bp fragments), and heterozygous for Lys/Gln . Similarly, in the case of Ala499Val, samples were classified as homozygous for Ala type (390 bp fragments), homozygous for Val type  and heterozygous for Ala/Val alleles  on the basis of banding pattern. Randomly selected 20% PCR products were sequenced by Sanger’s sequencing to confirm accuracy of the genotyping method.PCR generated amplicons of 493bp and 390 bp for Lys939Gln and Ala499Val loci, respectively. PCR products were digested without further purification with one unit of http://faculty.vassar.edu/lowry/VassarStats.html). Dominant, co-dominant, recessive and allelic genetic models were adopted for statistical analysis. Gene-environment interactions were assessed by the stratification of subjects on the basis of smoking and tobacco chewing status. P value < 0.05 was considered to be statistically significant.The distribution of genotypes was compared between cases and control using the Chi square test available at Vassarstats Online Calculator (www.ncbi.nlm.nih.gov/), EMBASE, and Google Scholar databases with the keywords, XPC and bladder cancer, XPC and carcinoma bladder, DNA repair gene polymorphisms, and carcinoma of bladder, XPC and UBC and bladder neoplasm in different combinations to identify the articles published up to November 2015. The search was limited only to the articles published in English. The articles thus retrieved were examined by reading the titles and abstracts, and full text articles of potentially relevant publications were further checked for their suitability for inclusion in this meta-analysis.We conducted a search in the PubMed . The combined odds ratio (OR) was estimated using the fixed effect (FE) model in the case of Pheterogeneity ≥ 0.10 and using the random-effects (RE) model in the case of Pheterogeneity < 0.10. High-resolution plots (forest plots) were generated to obtain the summary estimates. Publication bias was assessed from the distribution of studies on the funnel plot and statistically evaluated using Egger’s regression intercept test. Sensitivity analysis was conducted by removing the studies conducted on sample size less than 50 in either of the case control groups.Heterogeneity between the studies was assessed by the ‘Q’ statistic, which was considered statistically significant with P < 0.10. The heterogeneity was quantified by the IInclusion criteria comprised of the following: (i) Articles were original case–control or cohort studies on human subjects, (ii) The purpose of all the studies and statistical methods used were similar, and (iii) Articles had presented raw data necessary for the calculation of the crude odds ratio. Exclusion criteria comprised of the following: (i) Studies with obvious overlap of data with other included articles, (ii) Studies not providing enough information (raw data), and (iii) Studies not well described.Two authors analyzed the details of studies for meta-analysis. The following parameters from each study were recorded on a spreadsheet: the first author, the year of publication, the country of origin, ethnicity, and the number of cases and controls with genotype details.We have pooled data from our case-control study and other published studies on Ala499Val and Lys939Glnpolymorphisms. Meta-analysis was performed using the Comprehensive Meta-analysis software . Analysis was undertaken using dominant and recessive models. The strength of association was estimated by the OR and 95% CI.The results of a meta-analysis may be biased by the presence of systematic errors (bias) or random errors (play of chance) due to sparse data and repeated significance testing. Trials with low methodological quality, publication bias and small sample size may generate a false P-value. Therefore, we used a novel statistical analysis software, TSA  that calculates the required information size by adjusting the significance level for sparse data and repeated testing to confirm statistical reliability of the data in a meta-analysis29How to cite this article: Sankhwar, M. et al. Polymorphisms in the XPC gene affect urinary bladder cancer risk: a case-control study, meta-analyses and trial sequential analyses. Sci. Rep.6, 27018; doi: 10.1038/srep27018 (2016).


The CC genotype of T-786C was also associated with a higher risk of preeclampsia  than the CT + TT genotype. No association was found for VNTR 4b/a. Stratified analysis indicated that the increased risk was evident for high-quality studies both for G894T and T-786C, and for studies conducted among Caucasians and Africans for T-786C. However, the increased risk for T-786C among Africans needs further confirmation due to the high probability of false-positive reports. Our results suggested that G894T and T-786C polymorphisms, but not VNTR 4b/a, were associated with an increased risk of preeclampsia.Previous studies have examined the role of three NOS3 gene polymorphisms  in the susceptibility to preeclampsia with inconclusive findings. We therefore conducted an updated meta-analysis by including more studies. The most appropriate genetic model was chosen for each polymorphism by using a well-established method. Pooled results indicated that, compared with the GT + GG genotype, the TT genotype of G894T was associated with an increased risk of preeclampsia (odds ratio (OR) = 1.46; 95% confidence interval (CI) = 1.21–1.77, P < 0.001; I Preeclampsia is a clinical syndrome characterized by new-onset of hypertension and proteinuria after 20 weeks of gestation2in vitro and in vivo to modulate placental circulations, and the inhibition of NO production has caused preeclampsia-like syndromes in pregnant ratsl-arginine to l-citrullineAs a potent vasodilator, circulating nitric oxide (NO) plays a crucial role in endothelial function regulation, blood pressure control, and cardiovascular homeostasis, and NO is essential for a predisposition to preeclampsiaThe NOS3 gene is located on chromosome 7q35–36, with a length of 4.4  kbIn 2013, two meta-analyses on the associations between these three polymorphisms and preeclampsia risk were published but with inconsistent results1113141516171819202122A total of 791 articles were retrieved by a literature search . Of the 2428292 = 40.2%; Cochran Q = 41.83)  was 1.48 (P < 0.001); OR2 (GT vs. TT) was 1.11 (P = 0.037); and OR3 (GG vs. GT) was 1.44 (P < 0.001), respectively, suggesting that there is a strong recessive association (TT vs. GT + GG) for the putative susceptibility allele T with preeclampsia. The pooled OR and 95% CI for the association between G894T polymorphism and preeclampsia risk was 1.46  (P < 0.001) with moderate between-study heterogeneity (I= 41.83) , Fig. 2.2 = 46.9%; Cochran Q = 26.32). In the stratified analysis by ethnicity, the pooled estimate showed a significantly increased risk in the studies conducted among Caucasians  and among Africans . Similar to SNP G894T, when stratified by quality scores, an increased risk was observed only in high-quality studies with scores of 9 to 15   was 1.36 (P = 0.003); OR2 (CT vs. CC) was 1.06 (P = 0.392); and OR3 (TT vs. CT) was 1.26 (P = 0.029), respectively, also suggesting that there is a recessive association (CC vs. CT + TT) of the putative susceptibility allele C with preeclampsia. The overall fixed OR was significant , with moderate between-study heterogeneity (I= 0.003) , Fig. 2.For the VNTR 4b/a, OR1 (bb vs. aa) was 1.24(P = 0.193); OR2 (ab vs. aa) was 1.00 (P = 0.975); and OR3 (bb vs. ab) was1.07 (P = 0.690), respectively, suggesting that there is no association between the VNTR 4b/a polymorphism and preeclampsia risk. No more models were fitted for VNTR 4b/a.et al.et al.2decreasing from 40.2% to 36.0% or 24.9%.The sensitivity analyses indicated that no single study could change the pooled ORs obviously for both G894Tand T-786C . HoweverBoth Egger’s and Begg’s test revealed no significant publication bias, and the P values were 0.634 and 0.508 for G894T, and 0.141and 0.276 for T-786C. The funnel plots also indicated no evidence of publication bias both for G894T and T-786C .The false-positive report probability (FPRP) values for significant findings at different prior probability levels are shown in In this meta-analysis, we examined the associations between the three NOS3 gene polymorphisms  and the preeclampsia risk. Our results provide evidence of a significantly increased risk for preeclampsia for G894T and T-786C polymorphisms, under the recessive genetic model. No association was found between VNTR 4b/a and preeclampsia risk. In addition, stratification analysis showed that the association was more evident for high-quality studies both for G894T and T-786C, and among mixed Caucasian and African populations for T-786C. However, the results for Africans for T-786C need further confirmation due to the high probability of false-positive reports.et al.et al.2 = 50.0%), but no association was observed for T-786C and VNTR 4b/a. Another meta-analysis by Dai et al.2 = 44.2%) and that VNTR 4b/a was associated with a high risk in the recessive model . However, no association was observed for G894T .Recently, two meta-analyses were published focusing on NOS3 G894T, T-786C, and VNTR 4b/a polymorphisms and the preeclampsia risk11e.g., revealed in Dai et al.et al.et al.2411Our study, with the largest sample size, found a significantly increased risk of preeclampsia for G894T and T-786C polymorphisms, but not for VNTR 4b/a, which were partially consistent with the previous meta-analyses. In addition to the larger number of studies included leading to an increased statistical power, the following reasons could also explain the discrepancy. Firstly, the two previous meta-analyses did not exclude studies with controls not in HWE from the pooled analysis, and this may introduce potential selection bias in their findings. Not excluding studies with HWE-violation may result in the overestimation of the statistical significance of some postulated gene-disease associations, and it also seemed to modestly increase the between-study heterogeneity in some instances, thus distorting the findings14172829303132141821The evidence that NOS3 G894T and T-786C polymorphisms increase the risk of preeclampsia is biologically plausible. The NOS3 gene is responsible for encoding eNOS, a critical enzyme synthesizing NO through the conversion of L-arginine to L-citrulline in the vascular endothelium, by using molecular oxygen29In the stratified analysis by ethnicity for T-786C, a significantly increased risk of preeclampsia was found in studies conducted among Caucasians and Africans. The significant finding for Caucasians was probably due to the larger sample size in this subgroup. For Africans, the calculation of the FPRP values suggested that the possibility of false-positive reports could not be completely ruled out, and this still needs further confirmation. Stratified analyses also found significant results for high-quality studies both for G894T and T-786C polymorphisms, which may suggest an underestimation of the overall effects for these two polymorphisms because of suboptimal studies.a priori method to determine the most appropriate genetic model and calculated the FPRP to rule out the potential false-positive reports.One strength of our meta-analysis is a thorough literature search and review to identify as many studies as possible which are relevant to this topic. To the best of our knowledge, this meta-analysis included the largest number of original investigations in this area, thereby giving it the most sufficient study power. In addition, efforts were made to assess publication biases through Egger’s and Begg’s regression asymmetry test and funnel plot, and to pinpoint the potential sources of heterogeneity via stratified and sensitivity analyses. In addition, we used an e.g., the pooled sample sizes for the subgroup analyses among Asians and Africans for both G894T and T-786C were relatively small , and this may have attenuated the statistical power. Thus, more studies with larger sample sizes are needed to confirm the associations observed in these subgroup analyses. Finally, due to missing information about disease status , we cannot further explore the associations between NOS3 polymorphisms and preeclampsia risk by disease status, and this may also have influenced the interpretation.Several limitations of the present study also need to be taken into consideration. Firstly, moderate heterogeneity was found for G894T (40.2%) and T-786C (46.9%). For G894T, after excluding one of the studiesIn conclusion, this meta-analysis demonstrates that G894T and T-786C polymorphisms, but not VNTR 4b/a, in the NOS3 gene are associated with an increased risk of preeclampsia. The risk is more evident in high-quality studies both for polymorphisms and in studies conducted among Caucasians for T-786C. The risk for the T-786C polymorphism among Africans needs to be further confirmed. More high-quality studies should be conducted to establish the evidence of the associations between the NO synthase 3 gene polymorphisms and the preeclampsia risk for specific ethnic groups and by disease status.http://www.cnki.net) up to 24 November 2015 was performed. The following search terms were used: (“pregnancy induced hypertension” or pre-eclampsia or preeclampsia or pre eclampsia) and (“nitric oxide synthase” or eNOS or NOS3) and . No language restriction was applied; non-English articles were translated if necessary. We also scanned the reference lists of retrieved studies and review articles  to identify all of the relevant studies on this topic that might have been missed in database searches. Related articles generated by Google Scholar (http://scholar.google.com) and PubMed were also retrieved.We followed the standard criteria in the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) guidelines to conduct and report this systematic reviewTwo reviewers (ZFF and ZS) identified articles eligible for further review by independently performing an initial screen of identified titles and abstracts independently. Studies were considered for inclusion if they met the following criteria: (1) they were a case–control study (including nested case-control studies); (2) they used preeclampsia as an end point; (3) they included only apparently healthy controls ; (4) they included at least one of the three polymorphisms: G894T, T-786C, and VNTR 4b/a; and (5) they provided SNP genotype data and odds ratios (ORs) and corresponding 95% CIs. Studies were excluded if genotype frequency data in the controls demonstrated a departure from HWE. For the studies with overlapping data, only the most relevant articles with the largest dataset were included in the final analysis. Articles were retained when either of the two reviewers believed that they should be retained.Data were extracted by using a pilot-tested data extraction form. The following data were extracted: article title, the first author’s name, year of publication, country of the study performed, ethnicity of the study population , number of cases and controls, mean age and gestational age at delivery (GAD) of cases and controls, inclusion and exclusion criteria of the study population, source of controls, preeclampsia diagnosis criteria, genotyping method, and the numbers of cases and controls with different genotypes. The ORs and corresponding 95% CIs, after controlling for the minimal and the maximal adjusted number of covariates, were also abstracted.The quality of each investigation was assessed by quality assessment criteria derived from a previously published meta-analysis of molecular association studieset al.The departure of frequencies from expectation under HWE was assessed by chi-square goodness-of-fit tests in controls for each study. The strength of association between the three polymorphisms and preeclampsia risk was assessed by calculating ORs with the corresponding 95% CIs. We used the method proposed by Thakkinstian 2 statistics. If no significant heterogeneity was found between the studies, the pooled OR was calculated by using the fixed effects model (the Mantel–Haenszel method)39We used the Chi square-based Q-test to assess between-study heterogeneity. The heterogeneity was also quantified with IHow to cite this article: Zeng, F. et al. Associations between nitric oxide synthase 3 gene polymorphisms and preeclampsia risk: a meta-analysis. Sci. Rep.6, 23407; doi: 10.1038/srep23407 (2016).


Hepatitis B virus (HBV) infection is a major risk factor for the development of hepatocellular carcinoma (HCC) in China. At present, there still are 9.3 million chronic HBV-infected Chinese. Numerous studies have explored the association between possible factors and hepatocellular carcinoma risk, however, the results remains inconsistent. Therefore, we did this pooled analysis so as to get a precise result. Here, we took the chronic HBV-infected Chinese as the object. We systematically searched for studies evaluating whether the proposed factors changed HCC risk in PubMed, Chinese National Knowledge Infrastructure, VIP database and Wanfang data. Odds ratios (OR) with 95% confidence intervals (CI) were calculated by Review Manager 5.0 and publication bias was determined by Begg’s test and Egger’s test. In total, 3165 cases and 10,896 controls from 27 studies were included in this meta-analysis. Our results showed that pooled OR with 95% CI for each of the factors investigated were: non-antiviral treatment 2.70 , high HBV DNA levels 2.61 , alcohol consumption 2.19 , a family history of HCC 3.58  and male gender 2.14 , respectively. Our meta-analysis supports that high HBV DNA levels, non-antiviral treatment, alcohol consumption, a family history of HCC and male gender contributed to the risk of hepatocellular carcinoma in chronic HBV-infected Chinese from currently available evidence. Given the high prevalence of the non-antiviral treatment and alcohol drinking, behavior interventions for the two factors should be tackled first. Liver cancer, which consists predominantly of HCC, is the fifth most common carcinoma and the third most common cause of tumor-related deaths worldwide, leading to approximately 500,000 deaths every year ,2,3. In Considering the scale of the public health problem posed by HCC, over the past few decades, some measures have also been introduced to reduce the aflatoxin content in food and to reduce the lyngbya toxins in drinking water in China ,7,8. HowSince 1992, as part of an immunization project in China, hepatitis B vaccine has been used to inoculate in all newborns to effectively block mother-to-infant HBV transmission. However, the HBV infection rate remains high, with HBsAg carry rate of 7.2% for those aged between 1 and 59 years and 9.3 million chronic HBV-infected Chinese ,11. WherPrevious studies showed the modifiable risk factors included persistent high level of HBV replication, smoking and the habitual use of alcohol, however the effects of these factors were controversial ,38,39,40Meta-analysis is the implementation of statistical methods for combining and contrasting results from different studies to reduce random error and identify patterns or relationships in the context of a variety of evidence. In this study, we performed this meta-analysis to identify the true associations between possible factors and HCC risk.The data reported in our manuscript were cited from published literature, and had been approved by the ethics committee of Zhejiang provincial center of disease control and prevention (No.zjcdc-2015-1).All articles were retrieved from the following databases: Pubmed, Chinese National Knowledge Infrastructure (CNKI), VIP database and Wanfang data. Searches, using the search field “Title/Abstract”, were performed via using the search terms (“Chinese” or “China”) and (“hepatocellular carcinoma” or “liver tumors” or “tumor of liver” or “liver neoplasms”) and (“hepatitis b” or “hepatitis b virus”) AND (“risk factor”) from PubMed. Searches, using the search field “Abstract”, were performed via using the above-mentioned search terms from CNKI, VIP and Wanfang data journals published between January 1980 and October 2014.The present study was carried out following Meta-analysis in PRISMA guidelines .Only primary studies were included in our search. All eligible articles were case-control or prospective studies between January 1980 and October 2014. Eligible research articles not captured by the research strategies detailed above were retrieved by bibliography searches.Studies were included in the meta-analysis provided that: (1) the article reported a case-control or prospective study and had been accepted for publication with full text available; (2) all cases and controls were diagnosed by histopathological biopsy, or other national diagnostic criteria existing at that time, and possible risk factors were reported; (3) the article reported on chronic HBV-infected Chinese population (HBsAg as a marker of chronic HBV infection); and (4) the data to calculate OR with 95% CI was reported.Studies were excluded from the meta-analysis when: (1) The article reported other forms of viral hepatitis (hepatitis C or D) as the etiological agent; (2) The article did not provide a workable value for the main variable.To decide whether an article should be included or excluded, two independent reviewers carried out an assessment using a standardized data extraction form designed by our group. Data were extracted from each study by two separate investigators, and data about aflatoxin were not extracted because meta-analysis in the aflatoxin has been done . The litDiscrepancies between the decisions of the two reviewers were discussed. If a consensus was not achieved, the decision was made by a third reviewer. Articles were examined to eliminate duplicate reports of the same research.The following information was extracted from all of the acquired studies: the numbers of patients in each group, the characteristics of each group at baseline , and the study type. Definition of main outcomes: HCC, liver cirrhosis, CHB chronic hepatitis B and chronic HBV carries were diagnosed by the guidelines at that time ,44.p ≤ 0.1 the random-effect model was employed, and when p > 0.1 the fixed effects model was employed. In this meta-analysis, subgroup analyses were used to more thoroughly investigate the associations between different risk factors and HBV-related HCC, and Begg’s test and Egger’s linear regression test were also used to examine publication bias [p > 0.10), and the results of the Begg’s test were also not statistically significant . Analyses were performed using the software Stata version 9.0  and Review Manager 5.0 . The OR was not pooled when the number of OR of the risk factor were less than 5. All of the P-values were two-sided.The OR with 95% CI was used as the main outcomes to measure efficacy. Meta-analysis was performed using either the fixed-effect or random-effect model, depending on the statistical heterogeneity among studies as evaluated by Cochran’s chi-square test . Statistion bias ,47. The The selection of studies for inclusion in the meta-analysis was shown in According to the inclusive and exclusive criteria, all articles were retrieved and carefully reviewed to assess the eligibility. Twenty-seven eligible studies were identified after screening of 1161. A total of 27 studies, including 3165 cases and 10,896 controls were included in our research. Among the twenty-seven studies, following categories were studied: alcohol consumption 2180 cases), smoking 1432 cases), non-antiviral treatment (1094 cases), high HBV DNA levels 3831 cases), HBeAg positive 1592 cases), family histories of HCC (685 cases) and gender  3085 cases). The details of all of the studies evaluated in this meta-analysis are shown in 32 cases,92 cases,0 cases, 85 cases.31 cases,The available sample size of each study varied greatly, ranging from 80 to 11,893 objects. The mean age was greatly variable and the percentage of females also differed greatly see . The effects of non-antiviral treatment , alcohol consumption , smoking , high HBV DNA levels , HBeAg positive , family histories of HCC  and gender   on the development of HCC were investigated in this analysis and the results are shown in The pooled OR with 95% CI for the seven factors investigated were: non-antiviral treatment 2.70 , high HBV DNA levels 2.61 , alcohol consumption 2.19 , a family history of HCC 3.58 , male 2.14 , smoking 1.54  and HBeAg positive 1.25 , respectively.p < 0.10), and the random effect method was therefore used to pool the results for these factors. The heterogeneity test indicated that the variation of study-specific OR were not statistically significant (p > 0.10), and the fixed effect method was therefore used to pool the results for the other factors, and the conclusions remained largely unchanged when the random effect method was used. The results of statistical analysis and calculation are shown in The heterogeneity test indicated that the variation of study-specific OR for alcohol consumption, smoking, high HBV DNA levels, HBeAg positive, were statistically significant  have 2–4 times the risk of HCC development. The data do lead us to believe that controlling alcohol consumption might lead to decreases in HCC for some chronically infected Chinese. Since the proportion of the population with one or more of these risk factors is high in China, professional health education should be enhanced to promote cognitive and behavioral changes to reduce these harmful factors in society, and thus speeding up the process of preventing and controlling HCC. There is a high prevalence of non-antiviral treatment and alcohol drinking ,38,51,52In addition, this study showed that HBeAg positivity did not affect HCC development, and the results were different from those observed in previously reported ,33. A poThis study also showed that smoking did not affect HCC development, and the findings were also different from those observed in previously reported research . After oIt has previously been reported that age was a risk factor for HBV-related HCC development ; howeverHeterogeneity in the variation of study-specific OR for high HBV DNA levels and positive HBeAg was determined. One possible reason was that some cases had received antiviral treatment in three studies and four studies ,25,34,35This study has several limitations: (1) Some observational studies, retrospective studies and nonrandomized designs are susceptible to various biases such as inappropriate selection of subjects. These biases could have influenced slightly the internal and external validity of this study; (2) Although 27 eligible studies were included in our analysis, the sample for subgroup analysis was limited which could have affected the results; (3) Baseline data of all eligible studies such as observation time did not match well, and this could have underestimated the results; (4) Due to the low possible publication rate for negative studies, publication bias existed in some subgroup analyses of possible risk; (5) As the development of HBV-related HCC may be caused by multiple factors simultaneously, the interaction of factors may have contributed to the results. Due to data limitations, this article did not analyze this interaction.Our meta-analysis supports that high HBV DNA levels, non-antiviral treatment, alcohol consumption, a family history of HCC and male gender contributed to the risk of hepatocellular carcinoma in chronic HBV-infected Chinese from currently available evidence. Given the high prevalence of non-antiviral treatment and alcohol drinking, behavior interventions for these two factors should be tackled first.