Tea is supposed to have chemopreventive effect against various cancers. However, the protective role of tea in prostate cancer is still controversial. The aim of this study is to elucidate the association between tea consumption and prostate cancer risk by meta-analysis.versus non/lowest tea consumption levels were pooled on the basis of random effect model or fixed effect model as appropriate. Stratified analyses on tea type, population and study design were also conducted.A total of 21 published articles were retrieved via both computerized searches and review of references. Estimates of OR/RR for highest No statistical significance was detected between tea consumption and prostate cancer risk in meta-analysis of all included studies (odds ratio (OR) = 0.86, 95% CI (0.69-1.04)). Furthermore, stratified analyses on population ; non-Asian, OR = 0.89, 95% CI (0.72-1.07)) and tea type ; black tea, OR = 0.88, 95% CI (0.73-1.02)) also yielded non-significant association. Only the case–control study subgroup demonstrated a borderline protective effect for tea consumption against prostate cancer ).Our analyses did not support the conclusion that tea consumption could reduce prostate cancer risk. Further epidemiology studies are needed. Prostate cancer remains one of the most common cancers afflicting men today. It is one of the most frequently diagnosed malignancies among men in the world . AlthougCamellia sinensis[Tea is generally consumed in the form of black (fermented), green (unfermented) and oolong , all of which originate from the leaves of the plant  sinensis. Cellula sinensis. Numerou sinensis-8. Howev sinensis suggeste sinensis,11. Even sinensis revealedWe initially identified publications in the PubMed database up to July 2013 using keywords ‘tea’ and ‘prostate cancer’. Additional studies were identified from references cited in retrieved articles. Eligible studies should fulfill all the following inclusion criteria: (1) studies evaluating the tea consumption and prostate cancer risk; (2) the consumption of the natural green tea product, not of green tea extracts or supplements, were recorded; (3) the outcome of interest should be an incidence of prostate cancer; (4) odds ratio (OR) or relative risk (RR) estimates with corresponding 95% CIs  were provided. In studies with overlapping patients or controls, the latest study with the largest sample size should be included.Two investigators independently extracted the name of the first author, the year of publication, the population in which the study was conducted, study design, adjusted effects estimates, exposure assessment and adjusted covariates. Disagreement was resolved by either discussion or the third-party resolution. Considering that prostate cancer was a rare disease, the RR was assumed approximately the same as OR, and the OR was used as the study outcome. If a study provided separate adjusted ORs for different tea types or age stratifications, we treated them as independent studies.versus non/lowest tea consumption levels were pooled, as only those with highest tea consumption level were considered as habitual consumer. Between-study heterogeneity across the eligible comparisons was measured using the chi-square-based Q test and I-square test [P <0.05. According to the significance of heterogeneity, the data from a single study were combined using either the fixed effect or random effect models [vs. non-Asian), type of tea (black vs. green) and study design (cohort vs. case–control) were stratified. The potential publication bias was assessed graphically by funnel plot and estimated statistically by both Begg’s and Egger’s test [P <0.05 was considered significant for publication bias. In the overall study, sensitivity analysis would be performed to evaluate the effect of a single study contributing to overall summary and the Duval and Tweedie non-parametric ‘trim and fill’ method would be used to account for publication bias if any publication bias appeared to exist [P values were two-sided.The adjusted ORs/RRs and the corresponding 95% CI for highest are test . Heterogt models ,15. In ar’s test . P <0.05to exist . All staA total of 21 published articles reporting tea consumption and risk of prostate cancer satisfied the inclusion criteria and were included in our meta-analysis -12,18-35The overall result, presented in Figure PBegg’s = 0.020, PEgger’s = 0.066). But adjustment with ‘trim and fill’ method only yielded subtle variation ), which indicated that the conclusion from the overall summary was still reliable even though potential publication bias existed.For publication bias, visual exploration of the Begg’s funnel plot revealed an asymmetry shape Figure . FurtherPBegg’s = 0.027) and green tea subgroup .In the subgroup analyses Table , the stuNumerous studies have indicated daily tea consumption could be a non-invasive, economical and valuable means to prevent cancer. However, our current meta-analysis found that there was no significant association between tea consumption and reduced prostate cancer risk. Compared with the previous meta-analysis, our study has appended eight additional articles published after 2010 that were not included in the previous meta-analysis, which would strengthen the current conclusion.Additionally, stratified analysis was performed according to population, tea type and study design in the studies to further verify our conclusion. Consistent with the overall summary, the most stratified analysis showed that there was no association between tea consumption and prostate cancer, except the summary of case–control studies which demonstrated a borderline inverse association between tea intake and the risk of prostate cancer. Also, it should be noted that both green tea and black tea consumption did not reduce prostate cancer risk, even though green tea was generally considered having a stronger anti-cancer effect.The non-significant findings regarding the effects of tea consumption on prostate cancer in our meta-analysis contradicted the results of previous laboratory studies. Molecular study in cell-culture system and animal xenograft model research showed that both green tea polyphenol and black tea extracts -40 couldAs is often the case with meta-analysis, several potential limitations in our study should also be taken into account. First, we did not attempt to include unpublished observations and studies with insufficient information to estimate an OR/RR, which probably would bias our results. Second, most included studies did not provided detail information on tea consumption duration or drinking pattern, for example, number of new batches brewed each day, quantity of tea leaves used per batch . These pIn summary, our current meta-analysis suggests that available evidences from this epidemiological study are insufficient to conclude that tea consumption could reduce the risk of prostate cancer. Further epidemiological studies are warranted to examine the relationships of tea and coffee with prostate cancer risk.CI: Confidence interval; OR: Odds ratio; RR: Relative risk.The authors declare that they have no competing interests.YL and ZH performed statistical analysis and wrote the manuscript; XW and QM performed literature search and stratified the data; JQ and XZ provided meaningful discussion key points; LX revised and edited the manuscript. All authors read and approved the final manuscript.


NOXA and MCL1 may modify the SCCHN risk associated with HPV16 seropositivity.The cooperation between phorbol 12-myristate 13-acetate induced protein 1 (NOXA) and myeloid cell leukemia 1 (MCL1) is critical in the intrinsic apoptotic pathway. Human papillomavirus 16 (HPV16), by inducing p53 and pRb-E2F degradation, may play an essential role in development of squamous cell carcinoma of the head and neck (SCCHN) through NOXA-MCL1 axis-mediated apoptosis. Therefore, genetic variants of NOXA  and MCL1  were genotyped for 380 cases and 335 frequency-matched cancer-free controls of non-Hispanic whites.HPV16 serology was obtained by immunoadsorption assay. Four functional SNPs in the promoter of Associations between the four polymorphisms and SCCHN risk were not significant, while we observed a significantly joint effect on SCCHN risk between the polymorphisms and HPV16 seropositivity. Notably, this effect modification was particularly pronounced for oropharyngeal cancer in subgroups including never smokers, never drinkers and younger subjects.NOXA and MCL1 may modify the risk of HPV16-associated oropharyngeal cancer. The further identification of population subgroups at higher risk provides evidence that HPV-targeting treatment may help benefit SCCHN. However, larger studies are needed to validate our findings.Our results suggested that polymorphisms of  NOXA or co-expression of MCL1 and oncogene MYC can significantly accelerate the development of tumors such as lymphoma or leukemia [NOXA and MCL1 may be extensively involved in the tumorigenesis of different origins. However, whether NOXA and MCL1 play a role in the etiology of head and neck cancer remains unclear.Apoptosis has been implicated in the development of various human cancers, including squamous cell carcinoma of the head and neck (SCCHN). Growing evidence suggests that suboptimal apoptotic capacity leads to failure in responding to unfavorable stimuli and eliminating potentially neoplastic clones, which may cause accumulation of cells with cancer-prone mutations and therefore result in cancer susceptibility . The Bclleukemia -12. ThisNOXA or MCL1 promoter and the risk of HPV-associated SCCHN.As the eighth most common cancer worldwide, SCCHN accounts for nearly 3% of all incident malignancies in the U.S. with approximately 24,000 cases of oral cavity cancer, 13,000 of oropharyngeal cancer, and 12,000 of laryngeal cancer ,15, and NOXA and MCL1 in different cancer cells. Inhibition of either E6 or E6-associated protein can trigger a slight increase of NOXA levels, despite inducing a prominent accumulation of p53, in HeLa human cervix cancer cell [NOXA and MCL1 contain an array of confirmed or putative transcription factor binding sites, such as p73, E2F1, and STAT response elements [NOXA and MCL1 can be transcription-regulated, we hypothesized that HPV infection may deregulate NOXA-MCL1 axis-mediated apoptosis and subsequently affect the risk of SCCHN.Cumulating evidence indicates that HPV E6/E7 may regulate the expression of cer cell . More recer cell  proved tcer cell . In addielements . Since bNOXA and MCL1 promoters, and aimed to determine whether these genetic variants can, independently or jointly with HPV16 seropositivity, influence the risk of SCCHN, especially for those arising at the oropharyngeal site.To test this hypothesis, we genotyped the putatively functional polymorphisms in the Details of this SCCHN case–control study population were previously described elsewhere . BrieflyControls were recruited from a pool of cancer-free subjects from the hospitals in Houston metropolitan area. In this cancer-free control pool, a short questionnaire was used to determine each individual’s willingness to participate in the study before he or she was interviewed and asked to provide demographic and epidemiologic information including age, sex, ethnicity, smoking history, and alcohol consumption. Exclusion criteria for the control group included having had cancer previously, having known immune symptoms, received blood transfusions within the last 6 months, or were currently receiving immunosuppressive therapy. Approximately 78% of eligible subjects responded to the survey. As a result, 335 cancer-free control individuals were selected from the pool of potential controls by frequency matching on age (±5 years), gender, ethnicity, and smoking and alcohol drinking status. These variables were further adjusted in later multivariable logistic regression analyses to control for any residual confounding.The “smoker” and “drinker” are defined in consistent with CDC report  and our http://www.ncbi.nlm.nih.gov/projects/SNP) build 131 and bioinformatics tool (http://manticore.niehs.nih.gov/snpfunc) were used to identify potentially functional SNPs in NOXA and MCL1 promoter regions. We first scanned for all reported common SNPs  that are predicted to affect the function of binding to transcription factors and located in the coding region or in the 5’-untranslated region known as the transcriptional regulatory region in the upstream of the start codon (from −2000 to +1), as well as for SNPs located in 3’-UTR in the downstream of coding region (+1 kb downstream) for potential miRNA binding sites; and then the linkage disequilibrium (LD) between pro-target SNPs was assessed by using the public HapMap SNP database (http://www.hapmap.org). As a result, of the 64 SNPs reported in NOXA and 91 SNPs reported in MCL1, there were no common non-synonymous SNPs located in the coding regions and no common SNPs located in the 3’-UTR that may potentially alter the miRNA-binding profile; finally, there were two SNPs in the NOXA promoter region  and two SNPs in the MCL1 promoter region  predicted to be potentially functional and selected for genotyping. Genomic DNA obtained from the buffy coat of whole blood samples was extracted by the Qiagen DNA blood mini kit . Genotyping of NOXA and MCL1 polymorphisms was performed by using the TaqMan assay, and the pre-designed probes were purchased from Applied Biosystems . The pre-designed SNP genotyping assay IDs are C_30385519_20 for rs9957673, C_27902210_10 for rs4558496, C_1545306_10 for rs9803935, and C_27471449_10 for rs3738485. PCRs were run in a final volume of 5 μl per reaction, containing 10 ng genomic DNA, TaqMan Universal Master Mix, 80 × SNP Genotyping Assay Mix, and DNAse-free water. Four blank and positive controls and three random repeated samples were included in each plate for the genotyping assay quality control. The samples were genotyped by using the ABI 7900HT Sequence Detection System and Prism SDS 2.3 software.The NCBI dbSNP database  and their 95% confidence intervals (CI) were calculated to evaluate the association of NOXA and MCL1 genotypes and HPV16 seropositivity with the risk of SCCHN in both univariate and multivariable logistic regression models. The joint effects of examined genotypes and HPV16 seropositivity on risk of SCCHN were evaluated, which were further assessed in subgroups stratified by tumor sites, age, smoking and drinking status. The “linkage disequilibrium” (LD) procedure in the HelixTree v7.3.0  was used to estimate the LD by means of LD coefficients (D’) and the r2. All of the statistical analyses were performed by using Statistical Analysis System software , and two-sided P value < 0.05 was considered statistically significant.The differences in distributions of selected variables, HPV16 serological status and P < 0.0001). HPV16 seropositivity was significantly associated with increased risk of oropharyngeal cancer  but not SCCHN at non-oropharyngeal sites .We originally recruited 380 eligible cases and 335 controls of non-Hispanic whites in this study, of these subjects, eight cases and twenty controls failed in the genotyping assays. Therefore, 372 cases and 315 controls were included in the final genotyping analysis. The demographic characteristics and distribution of known SCCHN risk factors of the study population are summarized in Table NOXA and MCL1 genotypes and their associations with risk of SCCHN are shown in Table P = 0.069 for rs9957673, P = 0.139 for rs4558496, P = 0.898 for rs9803935, and P = 0.871 for rs3738485). Because the LD evaluation for the SNPs in NOXA or MCL1 is unavailable in the HpaMap database, we calculated the LD values for the SNPs studied. The NOXA rs9957673 and NOXA rs4558496 were in low linkage , while the MCL1 rs9803935 and MCL1 rs3738485 were in high linkage . There was no significant association observed between the putatively functional SNPs and the risk of overall or subtypes of the SCCHN . However, we found that the variants of both NOXA and MCL1 genes modified the association between the HPV16 seropositivity and the risk of SCCHN.The distributions of NOXA and MCL1 on SCCHN risk. By using the group with the NOXA rs9957673 CC genotype and HPV16 seronegativity as reference, those with the CT/TT genotypes and HPV16 seronegativity did not show association with increased SCCHN risk ; however, those with the CC genotype and HPV16 seropositivity had a 2.4-fold increased SCCHN risk , and those with the CT/TT genotypes and HPV16 seropositivity had a 3.6-fold increased SCCHN risk . Similarly, compared with those with the NOXA rs4558496 TT genotype and HPV16 seronegativity, those with the TT genotype and HPV16 seropositivity had an OR of 2.7 , and those with both TG/GG genotypes and HPV16 seropositivity had an OR of 2.8 . For the two linked SNPs in MCL1 promoter, when subjects with the MCL1 rs9803935 GG/GT genotypes and HPV16 seronegativity as the reference group, those with the TT genotype and HPV16 seronegativity had an OR of 0.8 , those with the GG/GT genotypes and HPV16 seropositivity had an OR of 2.1 , and those with the TT genotype and HPV16 seropositivity had an OR of 8.1 . The similar results were also observed for MCL1 rs3738485 polymorphism  than among older subjects (aged > 58 year) . Although these data suggest a potential joint effect between HPV16 infection and NOXA or MCL1 polymorphisms, larger studies are still needed to validate our findings.In this study, we did not observe significant main effects of the SNPs in the promoter regions of XL, but with higher priority and affinity to MCL1 [NOXA and MCL1.The deregulation of apoptosis is known as an important cause of neoplasia. In the intrinsic apoptotic pathway, apoptosis is usually mediated by cooperation among proapoptotic members (such as NOXA) and antiapoptotic members (such as MCL1) of the Bcl-2 family proteins. Differing from other BH3-only members, such as PUMA, NOXA usually executes its proapoptotic function by exclusively binding to MCL1, A1 or Bcl- to MCL1 ,35. Stud to MCL1 -38, whicThe decrease of SCCHN incidence in recent years accompanied by decline of tobacco use has not been observed for all head and neck cancer sites. However, the results from the America and European cohort studies suggested that the oropharyngeal cancer incidence  has steadily increased and shown to be associated with the involvement of HPVs, particularly the high risk type 16 ,19,39-41NOXA and MCL1 are targets of p53 and E2F, and p53 can affect the expression of its instant downstream target NOXA by directly binding to the promoter response element. In addition, other common transcription factors, such as E2F and p73, may also transcriptively regulate the expression of NOXA as well as MCL1, through respective response elements in the promoter regions of NOXA or MCL1[NOXA and MCL1 may jointly affect apoptosis induction, accordingly altering susceptibility to SCCHN. In agreement with that, we did observe a pronounced modification effect of these genetic variants on the risk of SCCHN, especially oropharyngeal cancer, associated with HPV16 seropositivity.Interestingly, A or MCL1, therebyNOXA, RITA  induced apoptosis in multiple cancer cells containing HPV16 and substantially suppressed the growth of cervical carcinoma xenografts in vivo [NOXA. By disrupting pRb-E2F complexes, the expression of HPV16 E7 protein up-regulates the NOXA expression, whereas, the binding of E2F to the NOXA promoter results in a significant reduction of NOXA-mediated apoptosis [NOXA expression. In addition, HPV E6- or E7-transfection can significantly up-regulate the expression of MCL1, therefore promoting lung tumor cell progression through PI3K pathway [NOXA and MCL1 dramatically increased for SCCHN at the oropharyngeal (HPV-related) site but not at non-oropharyngeal (HPV-unrelated) sites. Although our study implied significant trends of effect modification, we did not have adequate power to detect the interaction.Some other studies provided evidence for such kind of joint effects. In a very recent study, by blocking HPV E6-mediated p53 degradation to activate the transcription of  in vivo . Not onlpoptosis , suggest pathway ,31. Thesp53 or MDM2, may have interactive effects on risk of oropharyngeal cancer, particularly in never smokers and never drinkers [NOXA and MCL1 variants on the risk associated with HPV16 seropositivity for oropharyngeal cancer, particularly in never smokers and never drinkers. In addition, when the subjects were dichotomized by the mean age of the controls, we observed more apparent joint effects in younger, than older subjects. Though the mechanism is unclear, many researchers recently documented an apparent increase of incidence of HPV-related oropharyngeal cancer, particularly in young adults, perhaps due to distinct changes in sexual behaviors [NOXA and MCL1 and risk of SCCHN, as well as the joint effects between the genetic variants and HPV16 infection in the etiology of SCCHN.In consistence with our previously published studies in which HPV16 and genetic variants of p53 pathway genes, such as drinkers ,28, we aehaviors ,44. To tNOXA and MCL1 in the development of SCCHN is still unclear. Thus future studies are needed to focus on how these polymorphisms affect functional changes of the two genes. Secondly, the HPV16 serology assay used in our study could test the pre-exposure status but could not define the accurate infected organs or determine if the viral exposure took place before or after tumor development. Therefore, with this uncertainty applied to both the cases and controls, possible false-negative HPV16 cases might result in misclassification of the HPV16 status. However, the use of serology assay allowed for the inclusion of a cancer-free control group. Additionally, the relative small sample size could not provide enough statistical power for further stratified analysis or interaction analysis; thus we cannot exclude the possibility that our results could be by chance. Finally, we cannot deduce similar conclusions with certainty to other ethnic populations, because our study included only non-Hispanic white participants.However, there are several limitations in our present study. Firstly, although our results suggest a potential effect on SCCHN risk associated with HPV16, the underlying mechanism of NOXA and MCL1 may modify the risk of SCCHN associated with HPV seropositive, especially the risk of oropharyngeal cancer among never smokers, never drinkers and younger individuals. Regardless of the mechanism, our results implied that a potential joint effect among NOXA, MCL1 variants and HPV16 seropositivity may contribute the SCCHN risk. The identification of population subgroups at higher risk provide evidence that HPV-targeting treatment may help benefit SCCHN. However, well-designed studies with larger sample sizes in different ethnic populations are needed to validate our findings.We found that putatively functional polymorphisms of The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/159/prepub


Cyclooxygenase-2 (COX-2) is an inducible enzyme converting arachidonic acid to prostaglandins and playing important roles in inflammatory diseases as well as tumor development. Previous studies investigating the association between COX-2 polymorphisms and colorectal cancer (CRC) risk reported conflicting results. We performed a meta-analysis of all available studies to explore this association.All studies published up to October 2013 on the association between COX-2 polymorphisms and CRC risk were identified by searching electronic databases PubMed, EMBASE, and Cochrane library. The association between COX-2 polymorphisms and CRC risk was assessed by odds ratios (ORs) together with their 95% confidence intervals (CIs).Ten studies with 6,774 cases and 9,772 controls were included for −1195A>G polymorphism, 13 studies including 6,807 cases and 10,052 controls were available for −765G>C polymorphism, and 8 studies containing 5,121 cases and 7,487 controls were included for 8473T>C polymorphism. With respect to −765G>C polymorphism, we did not find a significant association with CRC risk when all eligible studies were pooled into the meta-analysis. However, in subgroup analyses by ethnicity and cancer location, with a Bonferroni corrected alpha of 0.05/2, statistical significant increased CRC risk was found in the Asian populations  and rectum cancer patients . In subgroup analysis according to source of control, no significant association was detected. With respect to −1195A>G and 8473T>C polymorphisms, no significant association with CRC risk was demonstrated in the overall and subgroup analyses.The present meta-analysis suggests that the COX-2 −765G>C polymorphism may be a risk factor for CRC in Asians and rectum cancer patients. Further large and well-designed studies are needed to confirm this association. Colorectal cancer (CRC) is the second most commonly diagnosed cancer with over 1.2 million new cases and 608,700 deaths in 2008 Cyclooxygenase-2 (COX-2) is an inducible enzyme that converts arachidonic acid to prostaglandins, which are potent mediators of inflammation. Through the production of prostaglandins, COX-2 is widely regarded as pro-inflammatory factor which can be activated by cytokines, mitogens, and growth factors at both the transcriptional and post-transcriptional levels Over the last two decades, a number of molecular epidemiological studies have been conducted to investigate the association between COX-2 −765G>C, −1195G>A, and 8473T>C polymorphisms and CRC risk, but the results remain controversial and inconclusive. With respect to −765G>C polymorphism, a meta-analysis by Cao et al We conducted a comprehensive literature search in PubMed, Embase, and Cochrane library databases up to October 01, 2013 using the following search strategy:  and  and . There was no restriction on time period, sample size, population, language, or type of report. All eligible studies were retrieved and their references were checked for other relevant studies. The literature retrieval was performed in duplication by two independent reviewers (Qiliu Peng and Xue Qin). When multiple publications reported on the same or overlapping data, we chose the most recent or largest population. When a study reported the results on different subpopulations, we treated it as separate studies in the meta-analysis. The study was performed according to the proposal of Meta-analysis of Observational Studies in Epidemiology group (MOOSE) The following criteria were used to include published studies: (1) Case–control studies which evaluated the association between COX-2 polymorphisms and CRC risk; (2) had an odds ratio (OR) with 95% confidence interval (CI) or other available data for estimating OR (95% CI); and (3) control population did not contain malignant tumor patients. Studies were excluded if one of the following existed: (1) the design was based on family or sibling pairs; (2) the genotype frequency was not reported; (3) there was insufficient information for data extraction; and (4) conference abstracts, case reports, editorials, review articles, and letters.Two reviewers (Qiliu Peng and Xianjun Lao) independently reviewed and extracted data from all eligible studies. To ensure the accuracy of the extracted information, the two investigators checked the data extraction results and reached consensus on all of the data extracted. If different results were generated, they would check the data again and have a discussion to come to an agreement. If these two authors could not reach a consensus, another author (Xue Qin) was consulted to resolve the dispute and a final decision was made by the majority of the votes. Data extracted from eligible studies included the first author, year of publication, country of origin, ethnicity, genotyping method, matching criteria, source of control, CRC diagnosis criteria, total numbers of cases and controls and genotype frequencies of cases and controls. Ethnic backgrounds were categorized as Caucasian, and Asian. When a study did not state the ethnic descendent or if it was not possible to separate participants according to such phenotype, the group reported was termed as “mixed ethnicity”.p value less than 0.05 was considered significant. The association of COX-2 polymorphisms with CRC risk was assessed using additive models, recessive model, and dominant model.The strength of the association between COX-2 polymorphisms and CRC risk was measured by odds ratios (ORs) with 95% confidence intervals (CIs). The significance of the pooled OR was determined by Z test and a Q test and 2I statistics to assess the statistical heterogeneity among studies Q test was hP>0.1 and 2I<50%, indicating the absence of heterogeneity, then a fixed-effects model (the Mantel–Haenszel method) was used to estimate the summary ORs Heterogeneity in meta-analysis refers to the variation in study outcomes between different studies. We used the p values were two-sided. To ensure the reliability and the accuracy of the results, two authors entered the data into the statistical software programs independently with the same results.Sensitivity analysis was performed by sequential omission of individual studies. For each polymorphism, publication bias was evaluated using a funnel plot and Egger's regression asymmetry test Based on the search criteria, 19 studies relevant to the role of COX-2 polymorphisms on CRC susceptibility were identified. Five of these articles were excluded: two were based on family or sibling pairs h = 0.022; AG vs. AA: Ph = 0.004; GG+AG vs. AA: Ph<0.0001). To explore the sources of heterogeneity, we first performed subgroup analyses. Subgroup analyses by ethnicity, cancer location, and source of controls showed that the heterogeneity was still significant in Asian populations, hospital-based studies, colon cancer patients and rectum cancer patients. Subsequently, we performed meta-regression analysis to further identify the source of heterogeneity. Meta-regression analysis indicated that the HWE in controls was the major source which contributed to heterogeneity. When we excluded two HWE-violating studies h = 0.833; AG vs. AA: Ph = 0.118; GG+AG vs. AA: Ph = 0.196). However, the significance of the summary ORs for COX-2 −1195A>G polymorphism in different comparison models were not influenced by omitting the two studies , the significance of pooled ORs under all contrast models in both total population and subgroup analyses was not influenced excessively by omitting any single study (data were not shown).For −1195A>G polymorphism, sensitivity analysis was further performed by omitting those two studies P = 0.330 for GG vs. AA; P = 0.853 for AG vs. AA; P = 0.312 for recessive model GG vs. AG+AA; and P = 0.890 for dominant model GG+AG vs. AA), −765G>C , and 8473T>C  polymorphisms; Begg's funnel plot and Egger's test were performed to assess the publication bias of literatures in all comparison models. The shape of the funnel plot did not reveal any evidence of obvious asymmetry. Then, the Egger's test was used to provide statistical evidence of funnel plot symmetry. The results still did not suggest any evidence of publication bias in −1195A>G , which was in accordance with the previously published meta-analysis by Cao et al. This finding may be biologically plausible. Cyclooxygenases are central enzymes in the prostaglandin pathway that convert free arachidonic acid into the intermediate prostaglandin H2 which is the precursor of prostaglandins, prostacyclin, and thromboxanes. Currently, three COX isoenzymes were reported: COX-1, COX-2, and COX-3 In subgroup analysis by ethnicity, the COX-2 −765G>C polymorphism presented a risk factor for CRC in Asian populations, but not in Caucasians. The inconsistent data among the different ethnicities may indicate different effects of the COX-2 −765G>C polymorphism on CRC risk in different ethnic genetic backgrounds. Nevertheless, owing to the limited number of relevant studies among Asian population included in this meta-analysis, the observed positive association between COX-2 −765G>C polymorphism and CRC risk in Asians is likely to be caused by chance because study with small sample sizes may have insufficient statistical power to detect a slight effect or may have generated a fluctuated risk estimate. Currently there are only 4 studies on COX-2 −765G>C polymorphism and CRC risk among Asian population Studies have suggested that cancers of the rectum and colon might be distinct tumors because they have a differing prevalence with a difference in clinical presentation, prognosis and possibly in genetic and environmental epidemiology. Thus, the COX-2 −765G>C polymorphism might influence carcinogenesis of colorectal tissues in a site-specific manner. Therefore, we carried out subgroup analysis according to cancer location. Our results suggested a significant increased CRC risk in rectum cancer patients  but not in colon cancer subjects, which was consistent with the results of the large sample study by Markar et al. In our meta-analysis, several limitations should be acknowledged. First, in subgroup analyses by ethnicity and cancer location, the sample size of population was relatively small for subgroup analyses, which may lead to relatively weak power to detect the real relationship. Second, our results were based on unadjusted estimates. We did not perform the analysis adjusted for other covariates such as smoking, drinking, obesity, red meat consumption, and so on, because of the unavailable original data of the eligible studies.In conclusion, our meta-analysis provided a more precise estimation based on larger sample size compared with the individual studies and previous meta-analysis. Our study suggested that COX-2 −765G>C polymorphism might contribute to colorectal cancer risk, especially in Asian populations and the rectum cancer patients. In order to further verify our findings, large well designed epidemiological studies are warranted.Figure S1Flow diagram of included studies for this meta-analysis.(TIF)Click here for additional data file.Checklist S1(DOC)Click here for additional data file.


Angiotensin-converting enzyme (ACE) gene has been implicated in susceptibility to cancer, but a large number of studies have reported inconclusive results. The aim of this study is to assess the association between the I/D polymorphism in the ACE gene and cancer risk by meta-analysis.The insertion/deletion (I/D) polymorphism in the A search was performed in Pubmed database, Embase database, Chinese Biomedical (CBM) database, China National Knowledge Infrastructure (CNKI) database and Weipu database, covering all studies until August 31, 2010. Statistical analysis was performed by using Revman4.2 and STATA 10.0.A total of 25 case-control studies comprising 3914 cancer patients and 11391 controls were identified. No significant association was found between the I/D polymorphism and over all cancer risks . In the subgroup analysis by ethnicity, no significant association was found among Asians and Europeans for the comparison of DD+DI vs. II. In the subgroup analysis by cancer types, no significant associations were found among lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer for the comparison of DD+DI vs. II. Results from other comparative genetic models also indicated the lack of associations between this polymorphism and cancer risks.ACE D/I polymorphism might not contribute to the risk of cancer.This meta-analysis suggested that the  The angiotensin-converting enzyme (ACE), a key enzyme in the renin-angiotensin system, plays the important roles of regulating of blood pressure and serum electrolytes ,2. It haACE gene is located on chromosome 17q23, and many polymorphisms have been identified [ACE expression or activity in blood and tissues among individuals [The human entified . The mosentified . It is centified . The I/Dividuals -18. Howeividuals ,20. ConsACE gene and cancer risks. The search terms were used as follows: 'cancer or carcinoma' and 'ACE or angiotensin-converting enzyme' in combination with 'polymorphism or mutation or variant'. The languages were limited to English and Chinese. The following inclusion criteria were used in the meta-analysis: (1) the study should evaluate the I/D polymorphism in ACE gene and cancer risk, (2) the study should be a case-control design, (3) enough information had to be provided to calculate the odds ratio (OR) with 95% confidence interval (CI), (4) the distribution of genotypes in the control groups should be consistent with Hardy-Weinberg equilibrium (HWE). Accordingly, the following exclusion criteria were also used: (1) abstracts and reviews, (2) studies in which the genotype frequencies were not reported, (3) repeated or overlapped publications. For studies with the same case series by the same authors, the most recently published studies or studies with the largest numbers of subjects were included.We searched literatures in Pubmed database, Embase database, Chinese Biomedical database(CBM) database, Chinese National Knowledge Infrastructure(CNKI) database and Weipu database to identify articles that evaluated the associations between polymorphisms in Data were independently checked and extracted by two investigators. The following items were collected from each study: first author's name, year of publication, country of origin, ethnicity, genotyping methods, cancer type, total number of cases and controls, genotype distributions in cases and controls.2 Xtest. OR and 95% CI was used to assess the strength of the association between the I/D polymorphism and cancer risk. We calculated the OR and respective 95% CI by comparing the carriers of rare alleles with the wild homozygotes (DI+DD vs. II).For each case-control study, the HWE of genotypes in the control group was assessed by using Person's 2 Xbased Q- and 2- Istatistic. Heterogeneity was considered significant for P less than 0.10. The fixed-effects model and random-effects model were used to pool the results. When the P value of heterogeneity was greater than 0.10, the fixed-effects model was used, otherwise, the random-effects model was used, as it is more appropriate when heterogeneity is present. The significance of the pooled OR was determined by the Z-test and P less than 0.05 was considered as statistically significant. To evaluate the ethnicity-specific, cancer type-specific effects, subgroup analyses were performed by ethnic group  and cancer types. Subgroup analyses by ethnicity were preformed if one ethnic population included more than three case-control studies. Subgroup analysis by cancer type were preformed if one cancer type canteined three and more than three individual studies. Comparisons of other genetic models were also performed .Heterogeneity among studies was assessed by a Publication bias was investigated by using several methods. Visual inspection of asymmetry in funnel plots was carried out. The Begg's funnel plots and Egger's test were also used to statistically assess publication bias ,22. SensACE gene polymorphism. Thus, 36 articles were left for data extraction. Six articles were excluded for not reporting the usable data. One article reported four cohorts each and each cohort was considered as a separate case-control study [There were 486 results relevant to the search words in the selected databases Figure . After rol study -28, and ol study ,29,14,35ol study ,33, 14 ool study ,32,34,35ol study , 1 of Af2 Xwas 79.09 with 24 degrees of freedom and P < 0.00001 in a random-effects model. Additionally, I-square value is another index of the test of heterogeneity. In Figure Z value was 1.38 (P = 0.17). The results suggested that the variant D allele carriers (DI+DD) do not have a significant increased risk of cancer compared with those individuals without D allele (II). Summary of the results of other genetic comparisons are listed in Table As shown in Figure Europeans and Asians. In the subgroup analysis by cancer types   and the Egger's test did not show any evidence of publication bias . Considering the limitation of studies in each subgroup, the weak associations between this polymorphism and different cancers should be discussed. It is possible that these null associations may be due to chance because studies with small sample size may have insufficient statistical power to detect a slight effect. Considering the limited studies in each cancer type, our results should be explained with caution.Heterogeneity is one of the important issues when performing meta-analysis. We found that heterogeneity between studies existed in overall comparisons. After subgroup analysis by ethnicity and cancer types, the heterogeneity was effectively decreased or removed in Asians and some cancer types, suggesting that certain effects of genetic variants are cancer specific and ethnic specific. The stability of this meta-analysis was analyzed by sequentially excluding individual studies; our results indicated stability of results. Publication bias is another important issue which should also be discussed in meta-analysis. After evaluating the publication bias by Egger's funnel plots and Begg's test, we did not detect a publication bias, indicating the strength of the results.ACE D/I polymorphism, which can be partly explained by the chips which were used in the original studies. There is probably a lack of adequate coverage on chips which also identify insertion or deletion a 287-bp sequence. It is noted worthy, the SNP rs4343, which is considered as a good proxy in Caucasians for I/D variant (r2 > 0.80), have been studied by Illumina genotyping arrays [Cancers have been considered as genetic diseases, and many genetic variants are contributed to cancer risks -40. It ig arrays ,48. ThusWe have to mention a recently published study by Ruiter in 2011. They alACE D/I polymorphism and the result also indicated a gene in RAS may not contribute to cancer risk. Third, the result of publication bias is not significant; indicating the conclusion of this study may be unbiased.Some limitations of this study should be addressed. First, only published studies in Chinese and English which were included by the selected databases were included for data analysis, some potential studies which were included by other databases or published with other languages or unpublished could be missed. Second, due to lack of original data, we could not evaluate the potential interactions of gene-gene and gene-environment. Third, this meta-analysis included data from Europeans, Asians, African-American and Latino populations, so that, the results are applicable to only these ethnic groups. Fourth, it is reported that PCR amplification of ACE I/D polymorphism using only flanking primer pairs would misclassify 4-5% of the ID genotype as the DD genotype and a second PCR should performed to confirm the DD genotype. HoweverACE gene may not contribute to susceptibility to cancer. However, larger well-designed studies are warranted to validate these findings. Moreover, future studies should also investigate gene-gene and gene-environment interactions to better display the association between the polymorphisms in ACE gene and cancer risk.In summary, this meta-analysis suggests that the I/D polymorphism in the ACE gene may not contribute to susceptibility to cancer.These results suggest that the D/I polymorphism in The authors declare that they have no competing interests.HF designed the research. YGZ and JZ searched the publications, extracted the data and wrote the materials and methods, results, discussion. YD checked all data. ZPX, HLH and CT was responsible for data synthesis and helped designed the study's analytic strategy. JH wrote the introduction. JH and XBL edited the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/12/159/prepub


SCL6A4 gene has been the most extensively studied functional polymorphism in psychiatry, providing worldwide evidence of its importance in several disorders, such as OCD.Obsessive compulsive disorder (OCD) is a neuropsychiatric condition that has been shown to be heritable and for which a major gene effect has been reported based on segregation studies1SCL6A4), located on chromosome 17q12in vitro compared with the short form of the 5HTTLPRThe efficacy of selective serotonin reuptake inhibitors (SSRIs) in the treatment of OCD has lead to the hypothesis of a serotonergic dysfunction in this disorderet alet alet alSCL6A4 coding region has not revealed differences in OCD patients8et alA possible association between 5HTTLPR polymorphism and OCD has been studied by some research groups. Billett SCL6A4 gene should always be performed. Family-based association analysis confirmed negative linkage disequilibrium between the 5HTTLPR polymorphism and OCD. TDT and HRR analysis did not reveal preferential “l” allele transmission. These studies did not support McDougle’s initial hypothesiset alIn addition, many association studies have resulted in controversial results because population stratification cannot easily be avoided8et alNew evidence showing that some of the previous results may differ since the involvement of the 5HTTLPR (A/G) gene polymorphism modulating the 5HTTLPR, it may impose to new studies the need to assess this polymorphism. In addition, most association data come from case-control studies that do not assess population stratification instead of family based samples. The study presented in this issue by Tibrewal


MTHFR) gene is one of the main regulatory enzymes involved in folate metabolism, DNA synthesis and remethylation reactions. The influence of MTHFR variants on male infertility is not completely understood. The objective of this study was to analyze the distribution of the MTHFR C677T and A1298C variants using PCR-Restriction Fragment Length Polymorphism (RFLP) in a case group consisting of 344 men with unexplained reduced sperm counts compared to 617 ancestry-matched fertile or normozoospermic controls. The Chi square test was used to analyze the genotype distributions of MTHFR polymorphisms. Our data indicated a lack of association of the C677T variant with infertility. However, the homozygous (C/C) A1298C polymorphism of the MTHFR gene was present at a statistically high significance in severe oligozoospermia group compared with controls . The genotype distribution of the A1298C variants showed significant deviation from the expected Hardy-Weinberg equilibrium, suggesting that purifying selection may be acting on the 1298CC genotype. Further studies are necessary to determine the influence of the environment, especially the consumption of diet folate on sperm counts of men with different MTHFR variants.The methylenetetrahydrofolate reductase ( Infertility is estimated to affect 10–15% of couples, and roughly half of these cases are due to the male factor MTHFR). MTHFR is an important regulatory enzyme in folate and homocysteine metabolism, which is necessary for a number of key biological cellular mechanisms MTHFR polymorphisms is associated with hyperhomocysteinemia that is considered as a risk factor for many diseases, including infertility MTHFR is much higher in testis than in other major organs in the adult mouse, suggesting that it might play an important role in testicular function MTHFR gene, C677T (A→V) MTHFR C677T variant decreases MTHFR activity and increases the homocysteine level. Similarly the MTHFR A1298C variant also reduces enzyme activity but to a lesser degree than C677T MTHFR polymorphisms and human male infertility A possible candidate for genetic susceptibility to spermatogenic failure is the gene 5,10-Methylenetrahydrofolate reductase  and semen analysis. Patients with known causes of infertility including genetic factors , lifestyle factors  and clinical factors  were excluded from the study group. The control group consisted of 450 fertile men who had more than one child and 240 normozoospermic men, all aged from 30 to 55 years. We obtained written informed consent from each subject and the study protocol was approved by the Committee on Research Ethics of Institut Pasteur du Maroc.Genomic DNA was extracted from peripheral leukocytes using the standard phenol–chloroform method.Genotyping of SNP C677T in the MTHFR gene: The C677T (rs1801133) mutation was analyzed by polymerase chain reaction (PCR) of genomic DNA using the following primer pairs: 5′-TGAAGGAGAAGGTGTCTGCGGGA-3′ (forward) and 5′-AGGACGGTGCGGTGAGAGTG -3′ (reverse). These primers generate a 198 bp fragment. PCR amplification was carried out in a total volume of 10 µL containing approximately 50 ng of genomic DNA, 200 µmol/L dNTPs, 10 pmol of each primer, 1.5 mmol/L MgCl2, 0.5 U Taq polymerase and 1 µL of 10× PCR buffer. The PCR reaction profile was: predenaturation at 94°C for 5 min followed by denaturation at 94°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 30 s for 35 cycles, with a final extra extension at 72°C for 7 min. PCR amplicons were digested with restriction enzyme HinfI. Then the products of digestion were electrophoresed on a 3% agarose gel stained with ethidium bromide and visualized using ultraviolet illumination. The wild type homozygote (CC), heterozygote (CT) and mutant homozygote (TT) showed one band (198 bp), three bands  and two bands (175 and 23 bp), respectively, because the substitution creates a HinfI recognition sequence which digests the 198 bp into 175 and 23 fragments.Genotyping of SNP A1298C in the MTHFR gene: The A1298C (rs1801131) mutation was identified using the following primer pairs: 5′-CTTTGGGGAGCTGAAGGACTACTAC-3′ (forward) and 5′CACTTTGTGACCATTCCGGTTTG-3′ (reverse) using the same PCR conditions used for C677T mutation. The amplified fragment of 163 bp was digested with restriction enzyme MboII. The A1298C abolishes an MboII restriction site. Digestion of the 163 bp fragment of the 1298AA genotype  gives five fragments of 56, 31, 30, 28 and 18 bp, the 1298CC genotype (mutated) results in four fragments of 84, 31, 30 and 18 bp, whereas the 1298AC genotype (heterozygous) produce six fragments of 84, 56, 31, 30, 28 and 18 bp. The digested fragments were separated in a 3% agarose gel stained with ethidium bromide and visualized using ultraviolet illumination.P values<0.05 were taken as statistically significant.The allelic frequencies were calculated by direct counting the alleles. The observed genotype frequencies for fertile and infertile men were compared with each other using Hardy–Weinberg equation  using the Chi squared test. Comparisons of genotype, allele, combination genotype and haplotype frequencies between control and infertile men were done by Chi square test, all p-values were based upon two-tailed tests and Odds ratios (ORs), 95% confidence intervals (CIs) were carried out, MTHFR polymorphisms C677T and A1298C in 344 infertile and 450 control men using the PCR-RFLP method. The polymorphism distribution of C677T in the MTHFR gene is summarized in MTHFR genotypes distribution for the C677T polymorphism was in Hardy-Weinberg equilibrium in controls and all infertile groups. We also determined a possible association of the combined genotypes (CT+TT) and allele T carrier frequencies, but the difference was not statistically significant. In addition, after classifying the patients into different groups, allelic frequencies were not statistically significant between patient and control cohorts.We analyzed MTHFR A1298C polymorphism are presented in MTHFR A1298C polymorphism,  compared to the control samples (2.46%) Moreover, this genotype (CC) showed highest frequencies in the severe oligozoospermia group .The genotype and allele frequencies obtained for the We also investigated possible associations of different combinations of both polymorphisms between controls and patients . The hapMTHFR gene encodes key regulatory enzyme involved in folate metabolism, and genetic variants of this gene may predispose some men to reduced sperm counts MTHFR activity by 30% in heterozygotes (CT) and 70% in homozygotes (TT) MTHFR C677T and infertility, MTHFR C677T polymorphism is capable of causing male infertility susceptibility in Asians, but not in Caucasians Folates are group B vitamins that play essential roles in the synthesis of nucleic acids and in epigenetic regulation of gene expression through remethylation of homocysteine into methioninine MTHFR A1298C variant results in a glutamine to alanine change at codon 429 (exon 7) and is found in a regulatory region of the MTHFR enzyme. This variant results in a decrease MTHFR activity, which is more pronounced in the homozygous (CC) than in the heterozygous (AC) or wild type (AA) genotype MTHFR p.429A variant is not thermolabile, in vitro studies have shown that this variant has approximately 65% of wild-type enzymatic activity compared to that of 40% of wildtype conferred by the C677T variant MTHFR 1298 C genotype in the control population. Although a number of factors may be responsible for the observed deviation from the Hardy-Weinberg expectation, we suggest that our data are consistent with purifying selection impacting on the 1298CC genotype. Indeed, we observed a strong association between the 1298CC genotype and severe oligozoospermia  as compared to the control group. There is growing evidence to indicate that the 1298C variant may be a genetic risk factor for male infertility. A recent study on 151 men with idiopathic infertility and 140 healthy fertile controls of Indian origin concluded that the MTHFR 1298CC genotype is a genetic risk factor for idiopathic male infertility in an Indian population MTHFR 677T allele was detected The MTHFR variant 1298C and unexplained reduced sperm counts, at least in the Moroccan population. Further studies are necessary to determine the influence of the environment, especially the consumption of dietary folate, on sperm counts of men with different MTHFR variants. Since a significant proportion of men with severe oligozoospermia have been reported to have methylation anomalies in their mature sperm MTHFR status, dietary folate and methylation profiles and their co-relation in human sperm also merits further investigation.In conclusion our data suggest a link between the


XRCC1 polymorphisms have been implicated in bladder cancer risk, but individually published studies show inconsistent results. The aim of our study was to clarify the effects of XRCC1 variants on bladder cancer risk.The XRCC1 Arg194Trp and Arg399Gln polymorphisms and bladder cancer risk. Heterogeneity and publication bias were also evaluated.A systematic literature search up to September 13, 2012 was carried out in PubMed, EMBASE and Wanfang databases, and the references of retrieved articles were screened. Crude odds ratios with 95% confidence intervals were used to assess the associations between P = 0.001) and 1.10 , respectively. After excluding the studies that were not in Hardy–Weinberg equilibrium, the estimated pooled odds ratio still did not change at all.A total of 14 and 18 studies were eligible for meta-analyses of Arg194Trp and Arg399Gln, respectively. Regrouping was adopted in accordance with the most probable appropriate genetic models. No obvious heterogeneity between studies was found. For overall bladder cancer, the pooled odds ratios for Arg194Trp and Arg399Gln were 1.69 (95% confidence interval: 1.25 to 2.28; XRCC1 Arg194Trp and Arg399Gln polymorphisms may be associated with elevated bladder cancer risk.The meta-analysis results suggest that  Bladder cancer is an important health problem worldwide. It is the seventh most common malignancy in men and seventeenth in women . An estiDNA carrying essential heritable information must remain stable in order to undertake its key physiologic functions, but it is continually vulnerable to many types of endogenous and/or exogenous damage; thus, genetic alterations could accumulate and tumorigenesis may occur because of the damaged DNA. The DNA repair system plays a pivotal role in maintaining the genome integrity and stability through the reversal of DNA damage. If accumulated genetic alterations occurred in corresponding DNA repair genes, their reversal capacity could be damaged, possibly increasing the risk of cancer in carriers . A largeXRCC1), located on chromosome 19q13.2–13.3, with 33 kb in length, is an important component of base excision repair (BER) [XRCC1. When damage occurs, XRCC1 recruited by DNA glycosylases, acts as a platform by regulating and coordinating a whole list of BER proteins and single strand break repair (SSBR) machinery [XRCC1 gene reported in the dbSNP database (http://www.ncbi.nlm.nih.gov/SNP), two genetic changes including Arg194Trp on exon 6  and Arg399Gln on exon 10  are the most extensively studied. Many previous studies have been conducted to evaluate the associations of XRCC1 polymorphisms with bladder cancer risk. However, the results of these studies remain inconsistent and contradictory, partially because a single study may be too underpowered to detect a possible small effect of the polymorphism on bladder cancer, especially when the sample size is relatively small. Thus, to clarify the effect of XRCC1 variants (Arg194Trp and Arg399Gln) on bladder cancer risk, we performed a meta-analysis of all eligible studies.X-ray repair cross-complementing group 1 (ir (BER) . BER conachinery ,12. AlthXRCC1 or X-ray repair cross-complementation group 1) and  and (polymorphism or variation or variant or mutation or genotype or gene). There was no language restriction. We evaluated potentially relevant papers by checking their titles and abstracts and all the studies matching the eligible criteria were retrieved. Additional studies were identified by a manual search of the references from retrieved articles and reviews.We carried out a systematic literature search in EMBASE, PubMed and Wanfang databases, covering all the papers published from their inception to September 13, 2012, using the following key words:  had a case–control design or nested case–control design, (c) had sufficient data for calculating an odds ratio (OR) with 95% confidence interval (CI). If multiple publications from the same population were available, the most recent or largest study was eligible for inclusion in this meta-analysis.Studies included in the present meta-analysis had to meet all the following criteria: (a) evaluation of the Data were extracted independently by two authors using a predefined data collection form, with disagreements being resolved by consensus. For each study, the following information was collected: first author’s last name, publication year, the country in which the study was carried out, ethnicity, numbers of cases and controls, genotyping methods, genotypes, and allele frequency information.The quality of each study was independently appraised by the same two authors using the quality assessment criteria, which were modified on the basis of previously published meta-analysis of molecular association studies ,14. The XRCC1 Arg194Trp and Arg399Gln polymorphism and the risk of bladder cancer. If there was a statistical heterogeneity among studies, the combined ORs and 95% CIs were estimated by the DerSimonian and Laird method [HWE in cases and controls was examined again in our meta-analysis using the goodness-of-fit test (significant at the 0.05 level). The ORs and their 95% CIs were used to calculate and assess the strength of the association between d method  in a rand method  in a fixORs 1, 2, and 3  were calculated for the genotypes: 1) TT versus CC, 2) CT versus CC, and 3) TT versus CT for Arg194Trp; and 1) AA versus GG, 2) GA versus GG, and 3) AA versus GA for Arg399Gln, respectively. These pairwise differences were used to determine the most appropriate genetic model. If OR1 = OR3 ≠1 and OR2 = 1, a recessive model is implied. If OR1 = OR2 ≠ 1 and OR3 = 1, a dominant model is suggested. If OR2 =1/OR3 ≠ 1 and OR1 = 1, then a complete overdominant model is indicated. If OR1 > OR2 >1 and OR1 > OR3 >1, or if OR1 < OR2 <1 and OR1 < OR3 < , then a codominant model is suggested.2 score. Heterogeneity was considered significant if the P-value was <0.10. The value of I2 was used to assess the degree of heterogeneity .Homogeneity of ORs across studies was tested by a Chi-square-based Q statistic and the ISensitivity analysis was performed in which the meta-analysis estimates were computed after omission of every study in turn. Cumulative meta-analyses of associations for each SNP were also conducted through assortment of studies with publication time.P <0.05 was considered to be representative of a significant statistical publication bias. All of the statistical analyses were performed with STATA 11.0 , using two-sided P-values.Publication bias was assessed using Begg’s test (rank correlation method)  and EggeXRCC1 genetic polymorphisms with the risk of bladder cancer. Of the selected studies, 14 [Twenty studies were included in this meta-analysis on the associations of the dies, 14 -32 were dies, 14 -28,30-38For the Arg194Trp SNP, OR1, OR2, and OR3 were 1.835 (95%CI: 1.343 to 2.507), 1.026 (95%CI: 0.920 to 1.146), and 1.581 (95%CI: 1.154 to 2.165), respectively, suggesting a recessive effect of the putative susceptibility allele T. Thus, the original grouping was collapsed, and CC and CT were combined, in accordance with a recessive model, into a C carrier group, the latter of which was compared with the TT genotype group.For the Arg399Gln SNP, OR1, OR2, and OR3 were 0.958 (95%CI: 0.850 to 1.080), 1.095 (95%CI: 1.014 to 1.183), and 0.884 (95%CI: 0.785 to 0.997), respectively, indicating that a complete overdominant model was applicable, that is, heterozygotes are at higher risk of bladder cancer than either homozygotes (GG or AA).XRCC1 Arg194Trp polymorphism was associated with an increased risk for bladder cancer in all subjects . Similarly, the Arg399Gln polymorphism was also found to be significantly associated with increased risk of bladder cancer .As shown in Figures 2 values of heterogeneity were 0% and all P values were more than 0.10, indicating no statistically significant heterogeneity between studies .Cumulative meta-analyses of the two associations were also conducted via the assortment of studies by publication time. The 95% confidence intervals became increasingly narrower with increasing sample size, indicating that the precision of the estimates was progressively boosted by the continual addition of more cases (data not shown).P = 0.640 for Arg194Trp; P = 0.820 for Arg399Gln) or with Egger’s test .There was no evidence of significant publication bias either with the Begg’s test  and, thus, it may lack sufficient statistical power to detect the real association and may have generated a fluctuated risk estimate.The results of the present study are in contrast with a previous meta-analysis published in 2008 , which cXRCC1 is closely related to BER pathway coordination by interacting with most members of the BER short-patch pathway. SNP of XRCC1 may increase the risk of some types of cancer by damaging the interaction of XRCC1 with other enzymatic proteins and, consequently, altering DNA repair activity [XRCC1 polymorphisms are also reported to be associated with some other cancers. The previous three meta-analyses have confirmed that the Arg399Gln polymorphism is associated with risk of childhood acute lymphoblastic leukemia [et al. reported that the XRCC1 Arg194Trp polymorphism is associated with an increased lung cancer risk [et al. suggested that the Arg194Trp polymorphism may be associated with cervical cancer risk [Our findings have some biological plausibility. It is widely accepted that certain genetic variants associated with repair of DNA substantially increase the risk of cancer in carriers because of the alteration of BER functions . BER is activity ; this maleukemia , breast leukemia , and proleukemia . Dai et cer risk  and the cer risk . By contSeveral limitations of this meta-analysis should be mentioned. First, the eligibility criteria for the inclusion of subjects and sources of controls were different from each other. No guarantee could be made among all those eligible studies that there were no potential bladder cancer cases in the controls. Second, because of the lack of the individual original data, our results were just based on unadjusted estimates, and gene–gene and gene–environmental interactions were not addressed in this meta-analysis. Third, although the Begg’s test and Egger’s test did not reveal any evidence of obvious publication bias, some inevitable publication bias may exist, because only studies published in English and Chinese were included in our meta-analysis. Finally, as shown in Table XRCC1 (Arg194Trp and Arg399Gln) may contribute to bladder cancer development. Whether it could be applied to genotyping for clinical assessment requires large-scale population studies among different ethnicities and regions.In conclusion, despite some limitations, the results of our meta-analysis suggest that two polymorphisms in BER: base excision repair; CI: confidence interval; HWE: Hardy–Weinberg equilibrium; OR: odds ratio; SNP: single nucleotide polymorphism; XRCC1: X-ray repair cross-complementing group 1.The authors declare that they have no competing interests.LPX, YQM and XX developed the study concept and participated in its design, data extraction, statistical analysis, manuscript drafting and editing. YWL and HC participated in the literature research, manuscript drafting and editing. JW and ZHH participated in design and data extraction. XLX and YZ participated in manuscript drafting, editing and statistical analysis. All authors read and approved the final manuscript.Score of quality assessment.Click here for file


BDNF) and Alzheimer’s disease (AD) has been widely reported, but the results remain controversial.The association between polymorphisms rs6265 and rs2030324 in brain-derived neurotrophic factor , Wanfang Med Online and China Biology Medical literature database (CBM) was performed. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using fixed or random-effects models. We excluded the studies with OR>3.0 or OR<0.3 for sensitive analysis. Subgroup analysis by ethnicity, form of AD and gender was carried out. Meta-regression was conducted to explore the potential sources of between-study heterogeneity.29 articles with 7548 cases and 7334 controls concerning rs6265 and 22 articles with 5796 cases and 5706 controls concerning rs2030324 were included in this meta-analysis. The combined evidence suggested rs6265 contributing significantly to the increased risk of AD in females (codominant: fixed-effects model (FEM): OR = 1.13, 95% CI = 1.04–1.23; dominant: FEM: OR = 1.17, 95% CI = 1.05–1.31), especially for Caucasian females  and female late-onset Alzheimer’s disease (LOAD) patients . No evidence indicated an association between rs2030324 with AD in codominant (random-effects model (REM): OR = 1.06, 95% CI = 0.89–1.26) and dominant  models.This meta-analysis suggested A allele of rs6265 might increase the risk of AD in Caucasian females and female LOAD patients. In addition, no evidence indicated an association between rs2030324 with AD. Further studies are needed to confirm these results. Alzheimer’s disease (AD) is an age-associated neurodegenerative disorder characterized by progressive decline in cognitive function, which typically begins with deterioration in memory (APP), presenilin-1(PSEN1), presenilin-2(PSEN2) APOE) SORL1) The key pathological changes that observed in AD brain tissue are the accumulation of neuritic extracellular amyloid plaques and intracellular neurofibrillary tangles BDNF gene, such as rs11030104, rs16917204, rs7103411, rs6265 and rs2030324. However, only the last two polymorphisms have been widely studied, with no linkage disequilibrium (LD) between them. What’s more, these results are inconsistent and individual studies have relatively small power to confirm this association. For example, the G allele of rs6265 confers risk effect for AD in subjects of Japanese  BDNF and AD susceptibility.Brain-derived neurotrophic factor (BDNF), as a member of the neurotrophic family, plays an important role in the growth, development, differentiation and regeneration of various types of neurons in the central nervous system BDNF” and “polymorphism” or “mutation” or “variant”. We also reviewed the references of included articles to identify additional articles not captured by our database searches.A literature search was performed for available articles that were published in English or Chinese (up to November 2013) from the following databases: (1) Pubmed; (2) Web of Science; (3) China National Knowledge Infrastructure (CNKI); (4) Wanfang Med Online; (5) China Biology Medical literature database (CBM). The search used the following keywords: “Alzheimer’s disease” or “AD” and “brain-derived neurotrophic factor” or “BDNF gene and AD susceptibility; (2) AD were diagnosed according to NINCDS–ADRDA criteria, DSM-IV criteria or CERAD criteria; (3) genotype frequencies were reported in the articles or could be calculated or ORs and 95% CIs can be obtained; (4) the genotype frequencies of controls are consistent with Hardy-Weinberg equilibrium (HWE); (5) if one study from the same population had been published more than once, we choose the most recent or complete one; (6) English or Chinese language articles were included.Two investigators reviewed all relevant studies independently to determine whether an individual study was eligible for inclusion. If the two investigators disagreed about the eligibility of a study, a senior researcher was invited to the discussion. The inclusion criteria were as follows: (1) case-control or cohort study published as an original study to evaluate the association between rs6265 and rs2030324 polymorphisms in Two investigators extracted the data independently and reached a consensus on all items. Information extracted from each study was as follows: first author, publication year, country, ethnicity of studied population, diagnostic criteria for cases, sample size, genotype distributions, mean age and the percentage of male.2) analysis was used to test deviation from Hardy-Weinberg equilibrium (HWE) for the rs6265 and rs2030324 genotype distribution of BDNF gene in control groups, and P<0.05 was considered as departure from HWE. Pooled measure was used as the inverse variance weighted mean of the logarithm of odds ratio (OR) with 95% confidence intervals (CI) to evaluate the strength of the association of rs6265 and rs2030324 in BDNF gene with risk of AD. We conducted analysis for each polymorphism considering dominant , recessive  and codominant  models, respectively. I2 of Higgins and Thompson was used to describe heterogeneity among studies 2>50%) was found; otherwise, the fixed-effects model (FEM) was adopted, and Mantel-Haenszel was used to assess the fixed effects. For rs6265 and rs2030324, meta-regression with restricted maximum likelihood estimation was performed to explore the potentially important covariates that might exert substantial impacts on between-study heterogeneity. Specific genetic variants causally associated with common diseases would have small effects (risk ratios mostly <2.0) 2>50% as the criteria to evaluate the key studies with substantial impact on between-study heterogeneity The chi-square , 176 articles from Chinese databases . 64 articles were reviewed in full-text. Furthermore, we excluded 30 articles according to the inclusion criteria, and obtained 1 additional article through references review. Finally, 35 articles were included in this meta-analysis. For rs6265, data from 29 published articles For both of the two polymorphisms, no individual study has excessive influence on the pooled effect in any of dominant, recessive and codominant models (data not shown). Harbord’s test showed no publication bias for both two polymorphisms.For overall analysis, no significant association was found between A allele and risk of AD in codominant , dominant  and recessive model  for rs6265 polymorphism. No evidence indicated an association between rs2030324 with AD in codominant , dominant  and recessive  models. The results for overall analysis are showed in 2>50%) was found in codominant and dominant models considering the association of rs6265 and rs2030324 polymorphisms with AD. Univariate meta-regression with the covariates of publication year, ethnicity, diagnostic criteria, form of AD, age (ratio of mean age in case group to that in control group) and gender  for the above-mentioned polymorphisms showed that no covariates have significant effect on between-study heterogeneity.Evidence for heterogeneity , dominant  and recessive  models. After stratified by gender, a statistical significant association between rs6265 and risk of AD was observed for females in codominant  and dominant model , but not in recessive model . However, no association was detected for males in codominant model , dominant model  and recessive model . When stratified by ethnicity in females, for Caucasian females, the A allele was found contributing significantly to the increased risk of AD in codominant model  and dominant model , not in recessive model . With regard to Asian females, no association was detected in codominant model , dominant model  and recessive model . When stratified by gender in LOAD patients, the A allele was observed significantly associated with AD in female LOAD patients in codominant model  and dominant model , but not in recessive model . However, no association was found in male LOAD patients in any of the above-mentioned models. For rs2030324, in the subgroup analysis, for Caucasian, no association was found in codominant model , dominant model  and recessive model . With regard to Asians, there was no association between T allele and AD susceptibility in codominant model , dominant model  and recessive model . When stratified by gender, there was still no association between rs2030324 and AD both for females and males in codominant model , dominant model . The pooled results are summarized in 2<50%) was found in the codominant, dominant and recessive models, and the results for both two polymorphisms are consistent with the ones without sensitive analysis. Furthermore, the “leave one out” analysis was carried out when significant heterogeneity (I2>50%) was observed. After excluded key articles with substantial impact on between-study heterogeneity, the data showed rs6265 confers a risk effect for Caucasians in codominant model . With respect to rs2030324, the result indicated T allele was associated with EOAD patients in codominant  and dominant model .After excluding articles with OR>3.0 or OR<0.3, low heterogeneity  was found in the codominant, dominant and recessive models, and the results are consistent with the one before heterogeneity analysis for rs6265 and rs2030324, strongly identified the stability of our results. Moreover, no publication bias was found in any of the above-mentioned models for both two polymorphisms.Between-study heterogeneity is common in meta-analysis for genetic association studies The major strength of our meta-analysis is that the results were based on the large number of participators, allowing a much greater possibility of reaching definitive conclusions. Additionally, we conducted subgroup analyses to explore the potential sources of heterogeneity, and sensitivity analysis was carried out to ensure the stability of our results. However, our meta-analysis also had some limitations. Firstly, lack of the original data of included articles made it impracticable to excluded potential confounders completely, especially the confounding of age. Secondly, different diagnostic criteria may have possible influence on the diagnosis of AD.In conclusion, this meta-analysis suggested A allele of rs6265 might increase the risk of AD in Caucasian females and female LOAD patients. In addition, no evidence indicated an association between rs2030324 with AD. Since potential biases and confounders could not be ruled out completely in this study, further studies are needed to confirm these results.File S1This includes the files Search Strategy S1 and Tables S1 to S3. Search Strategy S1. Keywords of literature search for different database. Table S1. Genotype and allele distribution for rs2030324 polymorphism in female and other subgroups. Table S2. Subgroup analysis on the relation of BDNF gene rs6265 polymorphism with AD in dominant and recessive model. Table S3. Subgroup analysis on the relation of BDNF gene rs2030324 polymorphism with AD in dominant and recessive model.(DOC)Click here for additional data file.Checklist S1PRISMA checklist.(DOC)Click here for additional data file.


Three first-line antituberculosis drugs, isoniazid, rifampicin and pyrazinamide, may induce liver injury, especially isoniazid. This antituberculosis drug-induced liver injury (ATLI) ranges from a mild to severe form, and the associated mortality cases are not rare. In the past decade, many investigations have focused the association between drug-metabolising enzyme (DME) gene polymorphisms and risk for ATLI; however, these studies have yielded contradictory results.PubMed, EMBASE, ISI web of science and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. A meta-analysis was performed to examine the association between polymorphisms from 4 DME genes  and susceptibility to ATLI. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Heterogeneity among articles and their publication bias were also tested.38 studies involving 2,225 patients and 4,906 controls were included. Overall, significantly increased ATLI risk was associated with slow NAT2 genotype and GSTM1 null genotype when all studies were pooled into the meta-analysis. Significantly increased risk was also found for CYP2E1*1A in East Asians when stratified by ethnicity. However, no significant results were observed for GSTT1.Our results demonstrated that slow NAT2 genotype, CYP2E1*1A and GSTM1 null have a modest effect on genetic susceptibility to ATLI. Tuberculosis (TB) is an important infectious disease plaguing the developed and developing countries worldwide, with more than nine million new cases of active tuberculosis reported annually. Isoniazid (INH), rifampicin (RIF) and pyrazinamide (PZA) are all widely used as first-line multidrug therapy for TB. Anti-tuberculosis drug induced liver injury (ATLI), a common serious adverse drug reaction, is one of the most challenging clinical problems, cause of hospitalization and life-threatening events. ATLI can be fatal if therapy is not interrupted on time, and the subsequent adherence problem may cause treatment failure and, relapse or drug resistance Of the various antituberculosis regimens, isoniazid is the main drug to induce hepatotoxicity Over the past few years, considerable efforts have been devoted to exploring the relationships between the DME polymorphisms and ATLI among various populations. However, existing studies have yielded inconsistent results. These disparate findings may be due partly to insufficient power, false-positive results, and publication biases. Therefore, we performed a meta-analysis of the published studies to clarify this inconsistency and obtain summary risk estimates for the association of specific polymorphism in DME and risk of ATLI.Eligible literature published before the end of May 2012 were identified through a search of PubMed, EMBASE, ISI Web of Science and CNKI  without language restriction. Search term combinations were as follows: drug-metabolising enzymes, antitubercular agents, drug-induced hepatotoxicity, drug-induced liver injury, genetic polymorphism, arylamine N-acetyltransferase, glutathione-S-transferase, cytochrome 2E1, acetylator phenotype, genetic susceptibility. All reference lists from the main reports and relevant reviews were hand searched for additional eligible studies.Eligible studies had to meet all of the following criteria: (i) the studies were published in peer-reviewed journals and were independent studies using original data; (ii) the studies provided genotype distribution information of polymorphism in both cases and controls or odds ratio (OR) with its 95% confidence interval and P value; (iii) the studies investigated the DME polymorphism using either case-control or cohort design; (iv) the studies described the genotyping method, equipment, and protocols used or provided reference to them and (v) the studies used TB patients without ATLI as controls.For each study, the following data were extracted independently by two authors: first author’s surname, year of publication, diagnosis criterion, age, sex, ethnicity, genotyping method, total number of cases and controls and genotype frequency in cases and controls. The results were compared, and disagreements were discussed and resolved with consensus.The proposed risk genotypes are NAT2 slow acetylator , CYP2E1*1A/*1A (homozygous wild type c1/c1) and homozygous null GST genotype (GSTM1 null/null or GSTT1 null/null). Our primary analysis for measuring the overall effects of every DME was to compare the genotype distribution for ATLI patients against controls by the contrast of the above risk genotypes vs. other combined genotypes.Odds ratio (OR) with 95% confidence intervals (CIs) was used to assess the strength of association between gene polymorphism and ATLI risk. Cochran’s chi-square-based Q statistic test was performed in order to assess possible heterogeneity between the individual studies and thus to ensure that each group of studies was suitable for meta-analysis. ORs were pooled according to the method of DerSimonian and Laird that takes into account the variation between studies, and 95% CI were constructed using Woolf’s method The combined search yielded 412 references. Study selection process was shown in P(Z) = 0.08; P(Q) = 0.11]. When studies were stratified for ethnicity, significant risks were found among East Asians . However, no significant associations were detected among Indian, Caucasian and other ethnic populations (P(Z) = 0.12; P(Q) = 0.05] and for small studies was 1.17 .Meta-analysis revealed no statistically significant association between ATLI and CYP2E1 c1/c1 genotype . When stratifying for ethnicity, an OR of 3.32 , 2.96 , 6.64  and 5.24  resulted for the slow acetylator genotype, among East Asian, Indian, Middle Eastern and other ethnic population, respectively (P(Z) <10−5; P(Q) = 0.26] and for small studies of 3.92 .Overall, there was evidence of an association between the increased risk of ATLI and the variant when all eligible studies were pooled into the meta-analysis. Using random effect model, the summary OR of the NAT2 slow acetylator genotype for ATLI was 3.18  with statistically significant between-study heterogeneity. This analysis is based on pooling of data from a number of different ethnic populations. When stratifying for ethnicity, an OR of 1.55  resulted for null genotype among East Asians; while no significant association was found among Caucasian and Indian populations (P(Z) = 0.003; P(Q) = 0.005] in big studies, compared to 1.26  in small studies.For ATLI risk and the null genotype of GSTM1, our meta-analysis gave an overall OR of 1.43 . When stratifying for ethnicity, an OR of 0.96  and 2.92  resulted for null genotype, among East Asian and Indian populations, respectively (P(Z) = 0.76; P(Q) = 0.44] in big studies, 1.88  in small studies.The meta-analysis resulted in a statistically non-significant association between GSTT1 deficiency and ATLI. The overall OR was 1.07  for ATLI appeared in compared with individuals with the positive genotypes.The effect of each genotype of GSTs was independently assessed. The data on both null genotype of GSTs among cases and controls were available in five studies, which included 460 cases and 1006 controls. The interaction between GSTM1 null and GSTT1 null, for which an OR of 1.12 [95% CI: 0.86–1.48; Sensitivity analyses were performed to assess the influence of each individual study on the pooled OR by sequential removal of individual studies. The results suggested that no individual study significantly affected the pooled OR, thus suggesting that the results of this meta-analysis are stable.P = 0.001; P = 0.23), GSTM1  and GSTT1 polymorphism .A funnel plot of these 24 included studies concerning NAT2 and ATLI suggested a possibility of the preferential publication of positive findings in smaller studies  and GSTM1 (OR = 1.43). Slow acetylators not only acetylate isoniazid more slowly but also monoacetylhydrazine, the immediate precursor of the toxic intermediates, to the harmless diacetylhydrazine GST, as an important phase II detoxification enzyme, was correlated to the susceptibility of alcoholic liver disease and many cancers In interpreting the results, some limitations of this meta-analysis should be addressed. Firstly, lack of clarification regarding the exact criteria used for the diagnosis of ATLI, and the Hardy-Weinberg equilibrium status among controls in several studies may over inflate our results. Secondly, variation in the anti-TB drugs administered in the studies we analyzed limited the possibility of examining the association of DME with any specific drug. In these studies, multiple anti-TB drugs were utilized in some research, while some other adopts the single-drug therapy. Thirdly, our meta-analysis is based on unadjusted estimates, whereas a more precise analysis could be performed if individual data were available, which would allow for an adjustment estimate . Additionally, it should be noted that the studies included patients from several different ethnic groups, with an overrepresentation of East Asian patients  and an underrepresentation of individuals of Caucasian descent. Since allele frequencies may vary considerably between ethnic groups, careful consideration of the potential effect of population genetics on genotypic and phenotypic distribution is warranted, but the limited samples currently available have hampered this effort.As a result of the heterogeneity of medication used, the duration of illness in different samples, and the different racial groups, it is possible that we have underestimated the effect size of the gene-drug response association. Furthermore, none of the studies formally accounted for medication noncompliance, which is prevalent in patients with TB. Put simply, when a patient does not take the prescribed anti-TB drug, the measured effect size of gene-drug response association is assessed as zero, whereas the true effect of genotype on the phenotype is perhaps larger. Nevertheless, despite the potential underestimation of effect size produced by these uncontrolled factors, we were still able to detect a significant association between the NAT2, GSTM1 and CYP2E1 polymorphism and anti-TB drug response. It is suggested that patients with high-risk genotypes should have regular liver biochemical tests in the first few months following administration of anti-TB drugs. Tailoring the anti-TB regimen to patients according to individual genetic profiles is expected in the coming years.In summary, our meta-analysis indicates that CYP2E1, NAT2 and GSTM1 genetic variation is significantly associated with anti-tuberculosis drug-induced liver injury. Polymorphisms in these DME, such as NAT2*4, may be particularly important in predicting clinical response to anti-TB drug treatment. Furthermore, interaction between candidate genes and other risk factors, such as diet, alcohol consumption, smoking, existing liver disease and other comorbid diseases, should be explored to realize the modification effect of these extrinsic factors to the expression of different genotypes.Figure S1The flow chart of the included studies.(TIF)Click here for additional data file.Figure S2Begg’s funnel plot of NAT2 polymorphism and ATLI risk.(TIF)Click here for additional data file.Figure S3Begg’s funnel plot of CYP2E1 polymorphism and ATLI risk.(TIF)Click here for additional data file.Figure S4Begg’s funnel plot of GST M1 polymorphism and ATLI risk.(TIF)Click here for additional data file.Figure S5Begg’s funnel plot of GST T1 polymorphism and ATLI risk.(TIF)Click here for additional data file.Checklist S1(DOC)Click here for additional data file.


Background and Objective: Nonsyndromic cleft lip and/or palate (NSCL/P) is a complex disease associated with both genetic and environmental factors. One strategy for identifying of possible NSCL/P genetic causes is to evaluate polymorphic variants in genes involved in the craniofacial development.Design: We carried out a case-control analysis of 13 single nucleotide polymorphisms in 9 genes related to craniofacial development, including TBX1, PVRL1, MID1, RUNX2, TP63, TGF?3, MSX1, MYH9 and JAG2, in 367 patients with NSCL/P and 413 unaffected controls from Brazil to determine their association with NSCL/P.Results: Four out of 13 polymorphisms  were presented in our population. Comparisons of allele and genotype frequencies revealed that the G variant allele and the AG/GG genotypes of TBX1 rs28649236 occurred in a frequency significantly higher in controls than in the NSCL/P group . The frequencies of rs4819522, rs7940667 and rs1057744 minor alleles and genotypes were similar between control and NSCL/P group, without significant differences. No significant associations among cleft types and polymorphisms were observed.Conclusion: The study suggests for the first time evidences to an association of the G allele of TBX1 rs28649236 polymorphism and NSCL/P. Key words:Cleft lip, cleft palate, polymorphism, genetic. Cleft lip and/or palate (CL/P) is the most common congenital anomaly of the face, and its etiology, which involves both genetic and environmental factors, is highly complex and the molecular basis remains largely unknown ,2. CL/P In this study we investigated the contribution of selected polymorphic variants in genes related to craniofacial development as risk factors of NSCL/P. We selected the following genes: TP63, which is expressed in the epithelial cells during lip and palate formation and mutations are related to ectrodactyly, ectodermal dysplasia and cleft lip/palate syndrome MIM 604292) (292 (4), -SamplesThis study included 367 unrelated patients with NSCL/P recruited from the Center for Rehabilitation of Craniofacial Anomalies, Dental School, University of Alfenas, Brazil. All patients were carefully examined and screened for the presence of associated anomalies or syndromes by the team of the Center for Rehabilitation of Craniofacial Anomalies. The clefts were classified with the incisive foramen as reference, and divided in 3 groups: cleft lip only (CLO), cleft lip and palate (CLP), and cleft palate only (CPO). Controls were chosen among subjects admitted as inpatients in the Dental School of the same University with conditions unrelated to clefting disorders (n=413). The control group was matched by age, place of birth and ancestrality. In a recent study we characterized the ancestrality of those samples by using a panel of 40 insertion-deletion markers . The ave-GenotypingGenomic DNA was extracted from oral mucosa cells as previously described . Criteri-Statistical AnalysesThe distribution of the genotypes was evaluated by Hardy-Weinberg equilibrium test, and the frequency distribution of alleles and genotypes under unrestricted and dominant genetic models was compared using cross-tabulation and standard chi-square test. In unrestricted analysis, it was assumed that all 3 genotypes have different disease odds, whereas in dominant model the heterozygote and rare homozygote have the same disease odds. To estimate the risk of each factor in the occurrence of NSCL/P, odds ratios (OR) and respective 95% confidence intervals (95% CI) were calculated. Multiple comparisons were corrected using the Bonferroni method. The assessment of gene-gene interactions was carried out using multifactor dimensionality reduction (MDR), which identifies combinations of genotypes associated with risk for the disease. In this study, we used 10-fold cross validation and 1000-fold permutation testing.TBX1, rs7940667 of PVRL1 and rs1057744 of JAG2, demonstrated genotypic variations in our population. The genotype distributions were consistent with Hardy-Weinberg equilibrium at all loci except for rs28649236 in TBX1.From 13 polymorphisms, only 4 of them, including rs28649236 and rs4819522 of TBX1 rs28649236 polymorphism was significantly lower in NSCL/P patients than that in controls (p=0.0002), with an odds ratio of 0.41 (95% CI: 0.25-0.67) (TBX1 rs4819522 genotypes in a dominant genetic model (CC vs CT+TT) was different between groups, but this difference was not kept after Bonferroni correction  . The fre25-0.67) . Howeverrrection . Similarrrection . The freobserved . There wobserved . HoweverTBX1 rs28649236 and PVRL1 rs7940667 was the best two factor model with a testing accuracy of 0.52. The best three factor combination, TBX1 rs28649236, PVRL1 rs7940667 and TBX1 rs4819522, demonstrated the accuracy of 0.59 and the cross validation consistency of 7/10. However, no model was significant in predicting the individual risk for NSCL/P.Potential interactions among the genes were assessed using MDR analysis . The comSeveral evidences indicate that NSCL/P, as resulted of genetic heterogeneity, low penetrance and influence of a variety of environmental factors, is a genetically complex disease ,2. AlthoThe rs28649236 nonsynonymous polymorphism in TBX1 replaces an arginine by a glycine at amino acid position 8 (p.Arg8Gly) of the protein sequence. Although there is no information of effect of this polymorphism on protein function, by using bioinformatic analysis  we obserBorderline associations with NSCL/P were found with other two markers of this study. We have found that CT and TT individuals for TBX1 rs4819522 polymorphism have an increased risk of NSCL/P in our population. However, this association did not resist the adjustment for multiple comparisons of Bonferroni. One of the main issues in association studies is how to evaluate the significance of multiple testing of polymorphims. Bonferroni correction is commonly applied, but it is too stringent, whereas an alternative approach would be to use replication of a nominal probability in a second data set, which is less rigid. Since we did not have access to a second dataset, we applied the Bonferroni correction. As significant associations for this study required an adjusted p?0.004, the significance of TBX1 rs4819522 polymorphism was lost. We realize that this correction is a limitation and may lead to loss of significant findings, but in light of not having verification, this approach appeared to be the most efficacious. No previous studies assessed the correlation of this polymorphism with NSCL/P. Because of the marginal association, this candidate marker requires replication in other sample sets, and also should impel others to investigate such association in different populations.The first evidence in favor of a role for PVRL1 variants in NSCL/P was obtained by Sözen and collaborators . Those aOn examining epistatic interactions, our results showed no significant correlation between the combinations and the risk for NSCL/P. Thus, rs28649236 of TBX1 gene appears to contribute independently to a decreased NSCL/P risk. Finally, nine of the probably polymorphisms of the selected candidate genes were not confirmed in the Brazilian population. These results are not surprising since two of them  are invariable in practically all populations studied , and the others  have never been studied before.In conclusion, this study suggests an association between the G allele of TBX1 rs28649236 polymorphism and the development of NSCL/P in the Brazilian population, while that the associations of TBX1 rs4819522, PVRL1 rs7940667 and JAG2 rs1057744 with NSCL/P require further studies.