Background. Recent studies suggested that two common polymorphisms, miR-146a G>C and miR-196a2 C>T, may be associated with individual susceptibility to hepatocellular carcinoma (HCC). However, the results remain conflicting rather than conclusive. Object. The aim of this study was to assess the association between miR-146a G>C and miR-196a2 C>T polymorphisms and the risk of HCC. Methods. A meta-analysis of 17 studies (10938 cases and 11967 controls) was performed. Odds ratios and 95% confidence intervals were used to evaluate the strength of the association. Results. For miR-146a G>C, the variant genotypes were associated with a decreased risk of HCC . For miR-196a2 C>T, significant association was also observed . Conclusion. The two common polymorphisms miR-146a G>C and miR-196a2 C>T were associated with decreased HCC susceptibility, especially in Asian population. Liver cancer is the sixth most common cancer in the world, with 782,000 new cases diagnosed in 2012. Hepatocellular carcinoma (HCC) is its dominant histological type and accounts for 70–85% of primary liver cancer . ChronicMicroRNAs (miRNAs) are a large family of short noncoding and evolutionarily conserved RNAs (about 21–23 nucleotides) that function as negative gene regulators . They exTo date, several studies have investigated the association between the two polymorphisms miR-146a G>C and miR-196a2 C>T and hepatocellular carcinoma susceptibility. However, the results remain inconsistent rather than conclusive. In order to estimate the overall risk of miR-146a G>C and miR-196a2 C>T polymorphisms associated with hepatocellular carcinoma and to quantify the potential between study heterogeneity, we carried out a meta-analysis on all eligible case-control studies with a total of 10938 hepatocellular carcinoma cases and 11967 controls.We searched the electronic literature from PubMed, EMBASE, Cochrane Central Register of Controlled Trials, ScienceDirect, Chinese National Knowledge Infrastructure (CNKI) databases, and Wanfang databases for all relevant reports , using the search terms “miR-196a2 or microrna 196a2 or rs11614913 or miR-146a or microrna 146a or rs2910164,” “polymorphism or variant or SNP,” and “hepatocellular carcinoma or liver cancer or HCC.” Publication country and publication language were not restricted in our search. In addition, studies were identified by a manual search of the reference lists of original studies. Of the studies with the same or overlapping data published by the same investigators, the most recent or complete articles with the largest sample sizes were included. In our meta-analysis, studies had to meet the following criteria: (a) evaluated the correlation between SNPs miR-146a rs2910164 and/or miR-196a2 rs11614913 and susceptibility to hepatocellular carcinoma, (b) contained available genotype frequency for both cases and controls, and (c) used a case-control design. Studies were mainly excluded for the following reasons: (a) no control population, (b) duplicating the previous publication, and (c) not for human cancer research.Two of the authors (Zhaoming Wang and Lei Zhang) extracted data independently complying with the inclusion criteria after the concealment of titles, authors, journals, supporting organizations, and funds to avoid investigators' bias. In the present study, the following variables were collected for each study: the first author's last name, year of publication, country of origin, ethnicity, source of controls , genotyping method, and sample sizes of genotyped cases and controls. In the cases of conflicting evaluation, the two investigators checked the data and agreement was reached after a discussion. If disagreement still existed, senior investigator Jianmin Bian was invited to the discussion.Z-test and P < 0.05 was considered as statistically significant. The heterogeneity between the studies was assessed by Cochran's Q-test [P > 0.10 for the Q test, the summary of OR estimate of each study was calculated using a fixed-effects model (the Mantel-Haenszel method) [P values.For the control group of each study, the Hardy-Weinberg equilibrium (HWE) was calculated using a goodness-of-fit chi-square test. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association between the miR-146a rs2910164 and miR-196a2 rs11614913 polymorphism and susceptibility to HCC. The pooled ORs were performed for allele frequency comparison , codominant model , dominant model , and recessive model , respectively. The significance of pooled ORs was determined by s Q-test . If the  method) . Otherwi method) . Sensiti method) . All anaThere were 382 published articles relevant to the search terms . By chooP = 0.000; CC versus GG: OR = 0.780, 95% CI 0.700–0.869, and P = 0.000; GC/CC versus GG: OR = 0.865, 95% CI 0.787–0.952, and P = 0.003; CC versus GC/GG: OR = 0.835, 95% CI 0.774–0.901, and P = 0.000 .For miR-196a2 C>T, the results were shown in Q-test was used in all of the genetic models to test heterogeneity. For miR-146a G>C, it showed no significant heterogeneity between studies during overall comparisons . Using metaregression analysis, none of them could explain the significant heterogeneity (Ph = 0.007 and I2 = 60.6%), source of controls (Ph = 0.009 and I2 = 61.0%), and sample size (Ph = 0.023 and I2 = 68.5%).parisons . For miRparisons : T versuogeneity . In addiTo assess the influence of each individual study on the pooled ORs, the sensitivity analysis was performed by removing a single study from meta-analysis sequentially. The results indicated that no single study influenced the pooled OR qualitatively . It suggt = −2.00 and P = 0.074 for miR-146a G>C and t = 1.18 and P = 0.273 for miR-196a2 C>T).The Begg funnel plot and Egger's test were conducted to assess publication bias. The shapes of the funnel plots did not reveal any evidence of obvious asymmetry in all comparison models . Then, EmiRNAs are involved in a variety of biological processes and regulate hundreds of gene targets . The stuMiR-146a G>C, or rs2910164 polymorphism which locates in the passenger strand of miR-146a, can disturb the secondary structure and maturation of miR-146a , 44. Xu Q-test and Egger's test showed no significant heterogeneity or publication bias, which indicated that our results were stable. Since miR-146a regulates hundreds of downstream gene targets, it is biologically plausible that rs2910164 polymorphism may alter the oncogenesis genetic pathway and modulate hepatocellular carcinoma risk.To explain these conflicting results, our meta-analysis, which was based on eleven studies and involved 5689 cases and 6790 controls, was conducted to derive a more precise estimation of the association. Our results suggested that miR-146a G>C polymorphism was associated with decreased risk of hepatocellular carcinoma among the included studies, especially in Asian population. Cochran's MiR-196a2 C>T polymorphism is another potential SNP in relevance to HCC. It not only affected the maturation of miR-196a2 but also could enhance the cell response to mutagen challenge , 45, 46.Our meta-analysis included 9 case-control studies to assess the relationship between MiR-196a2 C>T polymorphism and HCC. The results indicated that T allele carriers had significantly lower HCC susceptibility, especially in Asian population. Identification of heterogeneity is one of the most important goals of meta-analysis and heterogeneity existed in our study. However, through subgroup analysis and meta-regression analysis we could not find the source of heterogeneity, which suggested that these included studies may be different in either clinical, methodological, or statistical components and the quantitative synthesis.There are three similar meta-analyses about the association between the miR-146a G>C polymorphism or the miR-196a2 C>T polymorphism and the risk of hepatocellular carcinoma, but their studies showed different results from ours. Wang et al. 2012  carried There are some limitations in this meta-analysis that must be addressed. First, in the subgroup analyses, only one study originated from Caucasian, the size of which was small, and there was no African population. So our study mainly suggested the association between the two SNPs and HCC susceptibility in Asian population and may not be generalized to other ethnicities. Further studies on other ethnicities are necessary to validate the results. Second, lack of original data like HBV infection status, alcohol consumption, age, and gender from the included studies limited our further stratified analysis. HBV is one the most important risk factors to HCC , and theIn conclusion, our meta-analysis provided evidence that the two common polymorphisms miR-146a G>C and miR-196a2 C>T were associated with decreased HCC susceptibility, especially in Asian population. Additional well-designed, large studies are warranted to validate our findings and further functional studies should be conducted to elucidate its mechanism. More sufficient data such as hepatitis infection status, gene-environment interactions, and multiethnic groups should be considered in future studies to lead to a more comprehensive understanding of the association between miR-146a G>C and miR-196a2 C>T polymorphisms and the risk of HCC.


Colorectal cancer is one of the major causes of cancer mortality world-wide. Prevention would improve if at-risk subjects could be identified. The aim of this study was to characterise plasma protein biomarkers associated with the risk of colorectal cancer in samples collected prospectively, before the disease diagnosis.After an exploratory study on the comprehensive plasma proteome analysis by liquid chromatography-tandem mass spectrometry from ten colorectal cancer cases enrolled at diagnosis, and ten matched controls (Phase 1), a similar preliminary study was performed on prospective plasma samples from ten colorectal cancer cases, enrolled years before disease development, and ten matched controls identified in a nested case–control study within the Florence cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC) study (Phase 2); in Phase 3 the validation of the candidate biomarkers by targeted proteomics on 48 colorectal cancer cases and 48 matched controls from the Florence-EPIC cohort, and the evaluation of the disease risk were performed.Systems biology tools indicated that both in the Phase 1 and Phase 2 studies circulating protein levels differing in cases more than 1.5 times from controls, were involved in inflammation and/or immune response. Eight proteins including apolipoprotein C-II, complement C4-B, complement component C9, clusterin, alpha-2-HS-glycoprotein, mannan-binding lectin serine-protease, mannose-binding protein C, and N-acetylmuramoyl-L-alanine amidase were selected as promising candidate biomarkers. Targeted proteomics of the selected proteins in the EPIC samples showed significantly higher clusterin levels in cases than controls, but only in men . The remaining proteins were unchanged. Using multivariate logistic models a significant positive association emerged for clusterin, with an 80% increase in the colorectal cancer risk with protein’s unit increase, but only in men.The results show that plasma proteins can be altered years before colorectal cancer detection. The high circulating clusterin in pre-diagnostic samples suggests this biomarker can improve the identification of people at risk of colorectal cancer and might help in designing preventive interventions.The online version of this article (doi:10.1186/s12885-015-1058-7) contains supplementary material, which is available to authorized users. Colorectal cancer (CRC) is one of the main causes of death from cancer world-wide, with higher incidence and mortality rates in developed countries; it is more frequent in men than women .Most sporadic CRCs develop from a normal epithelium which, after a number of genetic and epigenetic molecular alterations, can turn into adenoma, a benign precursor lesion that can proceed to a malignant tumour . Though Diet and lifestyle changes may be effective for primary prevention and screening programs have reduced cancer mortality, but CRC continues to account for more than 9% of all new cancers ,3. PreveMass spectrometry-based proteomics offers a means of discovering robust disease biomarkers and this approach is increasingly used in cancer research. Several CRC proteomic studies have analysed samples from experimental models or from human surgical specimens to identify differences in the protein profile induced by cancer  and refeSo far, serum or plasma protein biomarkers have been sought mostly in CRC case–control studies, using samples collected at the diagnosis, when the tumour was already developed, but this limits the predictive value of the biomarker -9. By coWe used a mass spectrometry-based proteomic approach to identify early biomarkers of CRC in human plasma, dividing the investigation into three phases: first, in an exploratory study with a case–control design we compared the comprehensive plasma proteome from ten CRC cases enrolled at diagnosis, and ten age- and sex-matched controls, and identified differential circulating proteins (disease biomarkers) by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS); second, we did a similar preliminary study with a nested case–control design to identify candidate predictive biomarkers in plasma from ten CRC cases, enrolled years before the disease developed, and ten age- and sex-matched controls identified in the frame of a nested case–control study on CRC carried out in the Florence cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC) study; in the third phase, we validated the identified candidate biomarkers by liquid chromatography-selected reaction monitoring-mass spectrometry (LC-SRM-MS) on each individual sample of a series of 48 CRC cases and 48 matched controls from the Florence-EPIC cohort, and used these data to estimate the disease risk.Phase 1: Exploratory study. In this phase we examined ten newly diagnosed CRC cases and ten age- and sex-matched controls, identified in a hospital-based case–control study on CRC ongoing in the metropolitan area of Florence in the period 2006–2009. All cases were recruited when admitted to the Surgery Departments of the main hospitals in the area. All cases had histologically confirmed adenocarcinoma of the colon-rectum. The controls were randomly selected from a series of healthy adults residing in the study area. The controls were matched to CRC cases by sex and age. The demographic characteristics of Phase 1 subjects are shown in Table Phase 2 and Phase 3: Nested case–control study in EPIC-Florence. The rationale and methods of the EPIC study have been described elsewhere [lsewhere . Brieflylsewhere . In the Table Sample preparation, protein separation, identification of proteins with different circulating levels by global proteome analysis, and relative quantitation of candidate biomarkers by targeted proteomics, are fully described in Supplementary Methods  was used to map the differentially expressed proteins into biological networks and for functional interpretation of the protein data. Functional and Pathway analyses are described in Supplementary Methods  were based on the average normalised spectral counts (3 replicate runs) of the proteins identified by LC-ESI-MS/MS. Proteins showing at least a 1.5-fold up or down change  were considered to have different levels.Partial Least Squares-Discriminating Analysis (PLS-DA) was applied to Phase 1 and Phase 2 protein spectral counts, to find proteins discriminating CRC cases form controls. We used Simca-P v13  for data analysis after Pareto normalization.Phase 3. Between-groups comparisons of the selected protein relative amounts obtained after LC-SRM-MS were computed on the mean of three analytical replicates using the non-parametric Mann–Whitney U test, two-tailed; biomarker validation was done by Receiver Operating Characteristic (ROC) curve analysis. We used the Prism software v6 , setting the significance at P <0.05.vs. never-smoker), BMI as dummy variable , waistline as dichotomous variable according to the WHO cut-off , education as dummy variable , and daily intake of fruit, vegetables, red meat, and alcohol as continuous variables (g/day). We also applied multivariate logistic models separately for men and women; in these analyses the models were also adjusted by age.The association between each protein and cancer status was evaluated in the whole series by separate multivariate logistic models stratified by case-set, i.e. pair of cases and controls matched for sex and age, and adjusted for potential confounders. Each protein was used as continuous or dichotomous  variable. We ran four different logistic models, the first three using each protein as continuous variable, and the last using each protein as dichotomous variable. The logistic models were adjusted by smoking, waistline and education (model 1); by smoking, BMI, and education (model 2); by smoking, waistline, education and daily intake of fruit, vegetables, red meat, and alcohol (model 3); by smoking, waistline, and education (model 4). Smoking status was included as dummy variable  statistical program. A P-value <0.05 was considered significant.Mass spectrometric analysis after 1DE separation led to the identification of 138 proteins common to CRC cases and controls. Of these, only 94, listed in Additional file 2(X) = 0.699; R2(Y) = 0.996; Q2 = 0.922  = 0.604; R2(Y) = 0.985; Q2 = 0.685. A number of proteins showed a good discriminatory ability between the two groups. These proteins had VIP values >1  showed that most of them were involved in the complement systems . Figure The global plasma proteome data of the Phase 2 EPIC samples showed only a few changes in circulating protein levels, so these results alone did not allow the selection of candidate biomarkers. However, our data as a whole suggested there were proteins deserving further analysis. So we took account of all the possible suggestions given by Phase 2 data. We considered at least one of the following inclusion criteria: (i) proteins with normalised spectral count coefficient of variance <35% and FC ≥1.5 or ≤ −1.5; (ii) proteins giving VIP > 1 after PLS-DA. More stringent criteria were not applied, so as to have a more inclusive list of candidate biomarkers. After preliminary LC-SRM-MS analyses (not shown), proteins giving unreliable results were discarded. We ended up with the eight proteins listed in Table Additional file Bars in Figure As shown in Additional file To establish whether the CLU levels found in this study were in agreement with previously reported data, we developed a method for absolute quantitative analysis. The method showed a linear response between 0.2 and 3.2 pmol CLU/sample (R = 0.999). The absolute plasma CLU concentration in the EPIC samples was 1.83 ± 0.5 nmol/mL. Plasma CLU was respectively 1.92 ± 0.57 and 1.75 ± 0.40 nmol/mL in CRC cases and controls . In the males, EPIC CRC cases had significantly higher CLU than controls . No difference was seen in women (1.88 ± 0.64 and 1.85 ± 0.36 nmol/mL respectively in CRC cases and controls).Tables No significant association emerged for the whole series or the females Tables , there wWe further validated CLU as a very early biomarker to distinguish CRC cases from controls by ROC analysis. The results showed a significant AUC of 0.7225  only in men. The most convenient cut-off generated a sensitivity of 95% and a specificity of 75%. The ROC curve is shown in Figure Global proteomics has a key role in the identification of potential cancer biomarkers and this approach has been extensively used to discover CRC biomarkers . The sepTo have predictive value, an ideal biomarker should be easy to measure and should detect the disease at a very early stage. Prospective studies are extremely important, since biomarkers can be discovered on samples collected years before the disease onset. Proteomics has seldom been employed to search for candidate biomarkers in plasma samples collected before CRC was diagnosed, and this sort of investigation has been reported only in women . To the The study was preceded by an unbiased comprehensive analysis of the plasma proteome in a limited group of CRC patients enrolled at diagnosis and their matched controls. We then compared the results with those from an analogous global proteome analysis on a subpopulation of individuals from the EPIC cohort. In this early phase of the study we were interest in the identification of common changes in circulating protein profiles. To this end the differential proteome analyses were done on pooled samples, as this may minimize individual and technical variability while still maintaining the possibility of identifying changes induced by the disease, with the assumption that changes observed in pools correspond to the average of the individual changes ,17.Plasma proteins identified after the depletion of some high-abundance ones were still in the high to medium abundance range . The iniProteins involved in the immune response also showed altered levels, in agreement with evidence that an immune response is involved in CRC in addition to inflammation .Plasma carbonic anhydrase 1 and peroxiredoxin-2 were lower in cases than controls, but because of their high abundance in red blood cells these proteins were not taken into account, since their presence in plasma might be due to haemolysis during blood collection .Even though some plasma proteins identified in the exploratory study are different from those reported in earlier studies, the biological processes in which they are involved are essentially the same ,30.Comparison of the global plasma proteome of the exploratory and the EPIC studies indicated that most of the proteins identified were present in both studies, though in the EPIC there were fewer changes in the circulating protein levels. This comes as no surprise if we consider that the EPIC samples were collected several years before CRC diagnosis. However, PLS-DA analysis clearly distinguished EPIC CRC cases from controls and several proteins contributed to this result (proteins with VIP >1). Moreover, MetaCore enrichment analysis on proteins with changed levels indicated that the complement system cascade was the most significant process involved in both studies.We validated proteins playing a major role in the separation of cases and controls in the EPIC cohort by targeted proteomics, a powerful technique allowing the quantitation of candidate biomarkers in complex mixtures across multiple samples with high selectivity and sensitivity . Using aTargeted proteomics did not confirm the differences observed after global proteome analysis. The discrepancy is possibly due to the different sensitivity of the two analytical technologies, SRM-MS being more sensitive than MS/MS. Considering the whole EPIC population of our study, targeted proteomics indicated that CLU was the only protein slightly higher in CRC than in controls, but the difference was of borderline significance. This is in agreement with what was observed after the plasma global proteome analysis in the Phase 2 EPIC cohort, CLU showing FC = 1.32. Unlike in previous reports, we did not see any increase in circulating CLU in the Phase 1 exploratory study, possibly because of the limited number of individuals enrolled ,35. ThisInterestingly, this study found that CLU was significantly higher in EPIC CRC males than in their matched controls. No such difference was seen in women. This was corroborated by further statistical analyses showing that the CLU ROC curve significantly distinguished male CRC cases from their matched controls. Furthermore, after multivariate adjustments, CLU was significantly associated with CRC only in men, with OR 1.8. This sex-related difference might not be a chance result, as other biomarker levels differ in men and women. We have previously shown in a large EPIC cohort that high circulating C-reactive protein, a marker of systemic inflammation, was related to colon cancer risk in men, but not in women . More reThe molecular basis for the sex difference is not known, but androgens might possibly be involved; an early study reported a higher incidence of chemically induced CRC in male than female rats, androgens being involved in this sex difference . In addiCLU is a chaperone ubiquitously expressed and involved in several physiological processes, but also in tumour growth and carcinogenesis ,42. CLU CLU has already been proposed as a diagnostic biomarker of CRC, based on analyses on samples collected at diagnosis ,35. ThisIt is not clear why plasma CLU increases in preclinical samples, but we can formulate some hypotheses. Since plasma samples were collected prospectively, the CLU changes might be related to the host response, rather than it being a cancer-derived biomarker. The intestine wall is protected by a mucosal barrier whose homeostasis is maintained by a multidimensional network, including commensal microbiota, host innate immunity and genetics . Loss ofThe mass spectrometry-based analysis of the global plasma proteome identified, with high confidence, proteins involved in inflammation and/or immune processes, in samples collected at CRC diagnosis and years before it. In this preliminary study, the key finding is the identification of CLU as an early biomarker of CRC, at least in men. The main strengths of the study are the prospective design and the specificity of the analytical methods used to identify and validate candidate biomarkers. Though the small sample size is a limitation, the increased circulating CLU in preclinical samples warrants further investigation in a larger cohort of subjects to confirm the results of this pilot study and to assess the specificity of CLU as a biomarker for CRC, since it might help in identifying preventive intervention strategies.APOC2, apoliprotein C-II; CLU, clusterin; CO4-B, complement C4-B; CO9, complement component C9; FETUA, alpha-2-HS-glycoprotein (Fetuin A); MASP2, mannan-binding lectin serine-protease; MBL2, mannose-binding protein C; PGRP2, N-acetylmuramoyl-L-alanine amidase.


TLR4 polymorphisms have been related to PCa risk, but the relationship between TLR4 genotypes and aggressive PCa risk has not been evaluated by any systematic reviews.Toll-like receptor 4 (TLR4) is one of the best known TLR members expressed on the surface of several leukocytes and tissue cells and has a key function in detecting pathogen and danger-associated molecular patterns. The role of TLR4 in the pathophysiology of several age-related diseases is also well recognized, such as prostate cancer (PCa). We performed a systematic review and meta-analysis of candidate-gene and genome-wide association studies analyzing this relationship and included only white population. Considering appropriate criteria, only nine studies were analyzed in the meta-analysis, including 3,937 aggressive PCa and 7,382 controls.TLR4 SNPs reported by at least four included studies under any inheritance model . Pooled estimates from another ten TLR4 SNPs reported by three studies also showed no significant association . Meta-regression revealed that study type was not a significant source of between-study heterogeneity.Using random effects model, no significant association was found in the ten TLR4 polymorphisms were not significantly associated with the risk of aggressive PCa. Prostate cancer (PCa) is the most common malignancy since 1984, the most frequently diagnosed cancer, and the second leading cause of cancer-related deaths in 2013 among men in the USA TLR4 gene may modify the signaling of the immune response, which in turn may have effects on the pathogenesis of PCa.Toll-like receptor 4 (TLR4) is an important pathogen recognition receptor involved in detection of lipopolysaccharide (LPS) of Gram-negative bacteria and other exogenous or endogenous ligands TLR4 SNPs and PCa TLR4 SNPs and the aggressive type of PCa. Thus, we conducted a systematic review and meta-analysis of all genetic epidemiologic association studies that have evaluated the relationship between TLR4 polymorphisms and risk of aggressive PCa. Both candidate-gene studies and GWASs were included. The primary research questions are: (1) is there an association between TLR4 SNPs and risk of aggressive PCa and if so, what is the size of the relationship? (2) what is the validity of the evidence of association between TLR4 polymorphisms and risk of aggressive PCa?Three recent meta-analyses have explored the association between The execution of each individual study was previously approved by the respective institution. This systematic review was performed at the study level without access to individual-level data, and therefore, institutional review board approval was not necessary. Informed consent was obtained from each participant before the start of each individual study.http://gateway.ovid.com/), EMBASE (http://www.embase.com), Science Citation Index (http://science.thomsonreuters.com/cgi-bin/jrnlst/jlsearch.cgi?PC=K), and Online Mendelian Inheritance in Man (http://www.ncbi.nlm.nih.gov/omim) databases for all genetic association studies published before February 2013, using combinations of the search terms “toll-like receptor 4,” OR “toll-like receptor 4 gene,” OR “TLR,” OR “TLR gene,” OR “TLR4,” OR “TLR4 gene,” AND “prostate cancer,” OR “prostatic neoplasms.” GWASs were searched using combinations of the search terms “genome-wide association study,” OR “GWAS,” AND “prostate cancer,” OR “prostatic neoplasms.” In addition, we manually searched the reference lists from reviews and original articles to retrieve other papers relevant to the topic. Where there was overlap in the study populations of published papers, only the largest study was included. No language restriction was placed on the literature search strategies. Unpublished findings were not identified.The study was performed using pre-specified research objectives, search strategy, study eligibility criteria, methods of data extraction, and statistical analyses. Relevant studies were identified by searching the MEDLINE  independently reviewed each published paper and extracted relevant information examining the associations between TLR4 genotypes and risk of aggressive PCa were calculated using random effects models. Random effects models are preferred to fixed effect models because of the differences in study designs and study populations 2 statistic 2 statistic. To explore the inheritance mode for the effect of TLR4 polymorphisms, we evaluated the following genotype contrasts : (1) a/a and A/a combined versus A/A (dominant model); (2) a/a versus A/a and A/A combined (recessive model); (3) a/a versus A/A and A/a versus A/A (co-dominant model); (4) the increment of one minor allele (additive model). The Hardy-Weinberg equilibrium (HWE) was assessed via χ2 test. We did not perform haplotype analysis because none of the previous studies performed haplotype analysis specific for these SNPs. Because most GWASs did not adjust for covariates, this meta-analysis reported unadjusted pooled results.Meta-analyses were performed for SNPs that were reported by at least three included studies. The pooled odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between P values were two-sided. QUANTO program (http://hydra.usc.edu/gxe/) was used to evaluate statistical power of the association between TLR4 polymorphisms and aggressive PCa.To evaluate the presence of publication bias, we examined the funnel plot, by plotting the reciprocal of the standard error of log OR versus the log OR, for symmetry. The Egger linear regression test was also performed to assess funnel plot's asymmetry TLR4 gene (n = 2).Using the pre-specified search methodology we retrieved forty relevant publications . After eWe contacted the authors of the remaining 13 relevant studies for necessary details, and authors of three of the GWASs A total of 3,937 aggressive PCa cases and 7,382 controls were included in this work. Six studies were candidate-gene studies TLR4 SNPs and aggressive PCa, seven studies assessed rs4986790 For the association between TLR4 SNPs had been evaluated by at least 4 included studies. The minor allele frequencies (MAF) between case and controls were shown in TLR4 SNPs were reported by 3 studies, including one SNP located on the promoter region (rs10759930), one SNP located on 5′UTR (rs10116253), two intronic SNPs (rs11536869 and rs5030717), one non-synonymous exonic SNP (rs4986791), and five SNPs located on 3′ UTR . The locations of the explored SNPs  are shown in P = 0.01–0.03) but were kept in the analysis because the HWE tests were not significant after correction for multiple tests.Ten TLR4 SNPs  were not associated with the risk of aggressive PCa regardless of the inheritance model used . For the other 9 SNPs, P values ranged from 0.2 to 0.77.Funnel plots were used to assess the relationship between the ten sive PCa . Using tP value ranged from 0.15 to 0.79 for the ten TLR4 SNPs).Random effects meta-regression was performed under dominant model. Different study type (candidate-gene studies vs. GWASs) was not a significant source of between-study heterogeneity  and risk of aggressive PCa in the pooled analysis. The non-significant findings may be attributable to (1) failure to adjust for the conventional risk factors of PCa, e.g. family history of PCa, (2) inability to assess the within-population heterogeneity or geographic variation, and (3) the studied TLR4 SNPs may be more closely related to non-aggressive PCa.In the current meta-analysis, none of the examined TLR4 SNPs and overall PCa. Jing et al. TLR4 SNPs (rs4986790 and rs4986791) and found that rs4986790 showed a protective effect on overall PCa under co-dominant and recessive models. However, the effect was not statistically significant after stratification by ethnicity. Another work by Zhang et al. TLR4 SNPs  and did not find significant associations with overall PCa. The pooled estimates of Zhang et al. were derived from one Asian study Three recent meta-analyses evaluated the association between TLR4 polymorphisms and PCa risk. This may be explained by different ethnicity, within-population heterogeneity, case and control selection, gene-gene interactions, and gene-environment interactions. Although most of the relevant medical centers were in the “catchment” area, Cheng and colleagues Previous candidate-gene studies and GWASs found inconsistent results for the association between TLR4 SNPs, the SNPs reported in this study were under the influence of publication bias because only SNPs explored in ≥3 studies were included. We were unable to include three other GWASs because the authors did not respond to our data request There were some limitations of this study. One of them is the possibility of publication bias. Though the funnel plots did not reveal obvious publication bias among most of TLR4 polymorphisms and aggressive PCa, which shows more clinical relevance. All the included studies were reasonably well-designed epidemiological studies. Genotyping was carried out “blind” to the disease status, and assessment of aggressive PCa was carried out “blind” to the genotypes. This study had sufficient power (>0.95) to detect a potential OR of aggressive PCa associated with a SNP of 1.20. This study presents the best available evidence on the relationship between TLR4 polymorphisms and risk of aggressive PCa.This study had some advantages. To the best of our knowledge, this is the first meta-analysis on TLR4 SNPs were significantly associated with risk of aggressive PCa under any mode of inheritance. Control selection, different ancestry, small statistical power in some studies, publication bias, gene-gene and gene-environment interactions, different genotyping approaches, and issues of multiple tests may contribute to the inconsistent findings in previous studies. Meta-regression revealed that different study type (candidate-gene studies vs. GWASs) was not a significant source of between-study heterogeneity. Large-scale and well-designed studies using population-based controls and more studies in each ethnic group are needed to confirm our findings.In conclusion, this study found that none of the examined Figure S1Funnel plot of TLR4 SNPs. Funnel plot displays the publication bias for each study (indicated as one dot) exploring the relation between TLR4 SNPs and aggressive prostate cancer. SNPs reported by at least four studies were shown here.(TIF)Click here for additional data file.Table S1Pooled estimated ORs and 95% CIs for the association of TLR4 SNPs in aggressive PCa risk.(DOC)Click here for additional data file.Checklist S1PRISMA checklist.(DOC)Click here for additional data file.


I2 = 34.1%, P = 0.024). Based on subgroup analyses, a significant protective effect was observed with soy foods/products , in Asian populations , and in case-control studies . Therefore, soy isoflavone consumption was significantly associated with a reduced risk of CRC risk, particularly with soy foods/products, in Asian populations, and in case-control studies. However, due to the limited number of studies, other factors may affect this association.Colorectal cancer (CRC) is one of the most predominant solid carcinomas in Western countries. However, there is conflicting information on the effects of soy isoflavone on CRC risk. Therefore, we performed a meta-analysis to assess the association between soy isoflavone consumption and CRC risk in humans using PubMed, Embase, Web of Science, and Cochrane Library databases. A total of 17 epidemiologic studies, which consisted of thirteen case-control and four prospective cohort studies, met the inclusion criteria. Our research findings revealed that soy isoflavone consumption reduced CRC risk (relative risk, RR: 0.78, 95% CI: 0.72–0.85;  Colorectal cancer (CRC) is the third most prevalent cancer worldwide and one of the most common solid carcinomas in Western countries356789The association between soy isoflavone consumption and CRC risk has been evaluated1113141513141516171819202122232425262728http://www.ncbi.nlm.nih.gov/pubmed/), Embase (http://www.embase.com/), Web of Science (http://wokinfo.com/), and Cochrane Library (http://www.thecochranelibrary.com/). The search strategy included terms for outcome  and exposure . We designed, implemented, and reported our meta-analysis based on epidemiological study guidelinesWe performed a literature search of relevant studies published through November, 2015 using PubMed  studies written in English with case-control or cohort design; (ii) original human clinical trials that evaluated the association between soy isoflavone consumption and CRC risk; and (iii) use of risk point estimates, e.g., odd ratio (OR), relative risk (RR), or hazard ratio (HR) estimates with 95% confidence intervals (CIs).The extracted data were the first author’s name, year of publication, cancer type, population and country, total number of cases, dietary assessment method, estimates of soy isoflavone intake, and RRs or ORs with 95% CIs. Five publications reported separate RRs for soy foods and soy isoflavones, five publications reported separate RRs for male and female participants, and two publications reported separate RRs for colon and rectal cancers. In these cases, RRs were extracted individually.We assessed the association between soy isoflavone consumption and CRC risk using the reported RRs. Soy isoflavones were defined as soy foods, soy products, isoflavones, tofu, soy milk, miso, natto, genistein, daidzein, and flavonols. When adjusted and crude RRs were provided, the most adjusted RRs were extracted.We used HR and OR to evaluate CRC risk. HR and OR were considered to be approximations to RR, because CRC is a rare outcome in humans. Pooled RRs and 95% CIs were estimated on the basis of the most adjusted RRs or ORs for the highest versus lowest soy isoflavone intake.I2 and Q statistics to assess possible homogeneity of RRs across studies, which is a quantitative measure of inconsistency among studiesWe used P ≤ 0.05. Egger’s and Begger’s regression models were used to evaluate potential publication biasP values were from two-sided statistical tests.Data analyses were performed with STATA version 13.0. Statistical significance was set at et al. study13141516171819202122232425262728The study selection process is graphically described in Thirteen studies assessed the association between soy product consumption and CRC risk, while nine studies evaluated the association between isoflavone consumption and CRC risk. Among them, six studies separately presented findings for men and women, and two studies separately reported results for risk of rectal and colon cancers. Twelve studies were conducted in Asia and five in non-Asia countries . Data frP = 0.024) with a heterogeneity value (I2) of 34.1% , study populations (Asian or non-Asian), anatomical subsite , gender , and soy isoflavone type (soy foods/products or soy isoflavones) among the studies, we further conducted subgroup analyses to determine the effect of these factors in our analyses . We obtaWe analyzed 17 epidemiological studies that assessed the association between soy isoflavone consumption and CRC risk in humans. The findings revealed that the consumption of soy isoflavones was associated with a 23% reduction in CRC risk. CRC is caused by environmental  and genetic factors37β (ER-β) expressionβ expression, but reduce ER-α expression in the colon of female ratsEpidemiological and animal studies have found that the consumption of dietary soy decreases the incidence of certain tumors, including those of the colon and rectum39404142Our meta-analysis had some limitations. First, only studies written in English were included. Second, most studies used FFQs as the main dietary assessment method. Recall bias may have affected the results. Additionally, it was challenging to predict the effect of misclassification of case-control studies on the results. Third, certain confounding factors were not adjusted in the evaluated studies, e.g., family history of CRC, smoking, and alcohol consumption, which are important risk factors of CRC4748P value > 0.05; the shape of the Begger’s funnel plot seemed symmetrical. There was no significant evidence for publication bias in our meta-analysis (P > 0.05).There was heterogeneity across the studies in terms of soy isoflavone consumption, which is not surprising considering the differences in the study designs, soy types, and gender. Additionally, differences in geographic area may have contributed to the heterogeneity results; most of the studies were conducted in Asia, where the consumption of soy is high. Moreover, while some studies were adjusted for age, gender, and family history of CRC in the calculation of risk estimates, not all parameters were considered. The measurement units varied among the studies. Sensitivity analysis was performed by sequentially omitting one single study to assess the effect of each study on the overall results . The EggP = 0.024) and an I2 value of 34.1%. A statistically significant protective effect was observed with soy foods/products , in Asian populations , and with case-control study designs . Soy isoflavones play an important protective role in the pathogenesis of CRC, by a mechanism that remains to be elucidated. More cohort and intervention studies are required.In summary, our meta-analysis provided an updated and comprehensive evaluation of the association between soy isoflavone consumption and CRC risk, with an RR value of 0.77 .


The association between fish consumption and rheumatoid arthritis (RA) is unclear. The aim of this paper was to summarize the available evidence on the association between fish consumption and risk of RA using a dose-response meta-analysis.Relevant studies were identified by a search of MEDLINE and EMBASE through December 2013, with no restrictions. A random-effects dose-response meta-analysis was conducted to combine study specific relative risks. Potential non-linear relation was investigated using restricted cubic splines. A stratified analysis was conducted by study design.Seven studies (four case-controls and three prospective cohorts) involving a total of 174 701 participants and 3346 cases were included in the meta-analysis. For each one serving per week increment in fish consumption, the relative risk (RR) of RA was 0.96  0.91 to 1.01). Results did not change when stratifying by study design. No heterogeneity or publication bias was observed. When fish consumption was modeled using restricted cubic splines, the risk of RA was 20 to 24% lower for 1 up to 3 servings per week of fish  as compared to never consumption.Results from this dose-response meta-analysis showed a non-statistically significant inverse association between fish consumption and RA. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that affects the joints, leading to cartilage and bone destruction. The worldwide prevalence of this chronic disease ranges between 0.5% and 1.0%  and alcoThe association of fish consumption with risk of developing RA is still unclear, because results from both case-control and cohort studies are mixed. One prospective cohort  and two The aim of the present study was to quantitatively summarize the published evidence from epidemiological studies on the association between fish consumption and RA using a dose-response meta-analysis.We conducted a literature search through December 2013 using PubMed and EMBASE databases. The term, rheumatoid arthritis, was used in combination with fish, or seafood. Reference lists from acquired articles were also examined. Studies were included in the meta-analysis if they met the following inclusion criteria: the exposure was fish or seafood consumption; the outcome was incident RA; relative risk (RR) or odds ratio (OR) estimates were reported with their 95% CI.From each study we collected information on the first author’s last name, publication year, country, study period, number of cases and controls or cohort size, gender and age of study participants, covariables adjusted for, and RRs or ORs with 95% CI for each exposure category. If multiple RRs and ORs were presented, we extracted the estimates from the maximally adjusted model in order to reduce the risk of possible unmeasured confounding. Data extraction was performed independently by two of the authors (DDG and AC).The quality of studies was assessed using the Newcastle-Ottawa quality assessment scale (NOQAS) for cohort and case-control studies, with which each study was judged based on the selection of the study groups, the comparability of the groups, and the ascertainment of exposure and outcome . The scoet al. [A two-stage dose-response random-effects meta-analysis was conducted to combine risk estimates -19. The et al. , the meaet al. .et al. was excluded from the flexible dose-response analysis because it reported information only on the linear trend [A potential non-linear relation between fish consumption and RA risk was investigated using restricted cubic splines with three knots at fixed percentiles  of the exposure distribution. Departure from linearity was assessed by testing the null hypothesis that the coefficient of the second spline was equal to zero . The stuar trend .Q-test and the I2 statistic [In all meta-regression models, statistical heterogeneity between studies was evaluated with the Cochran tatistic  that asstatistic . Sensititatistic .Of the 384 studies identified through PubMed and EMBASE, only seven examined the association between fish consumption and risk of RA and were included in this dose-response meta-analysis Figure . Charactet al. [et al. [Three studies showed a borderline statistically significant inverse association between total fish consumption and risk of RA ,9,10, whet al. , which, et al. . Broiled [et al. .Figure 2I2 = 0.0%, P-value =0.41) or of publication bias . Results stratified by study design were similar: the pooled RR was 0.96  and 0.97  for case-control and for cohort studies respectively. We also stratified by number of covariates considered: the pooled RR of the three studies that adjusted only for age and gender was 0.88 , while it was 0.97  for the four studies that considered also covariates other than age and gender. A sensitivity analysis excluding one study at the time showed that the linear trend ranged between 0.95  and 0.97 .We first assumed a linear-response model for the association between fish consumption and risk of RA. The risk of RA decreased by 4% for every increase of one fish serving per week, although this change was not statistically significant  Figure . There wnon-linearity =0.15)  Figure . HoweverThe present study is the first dose-response meta-analysis on the association between fish consumption and risk of developing RA. Results from this meta-analysis showed a weak inverse association between total fish consumption and risk of RA. The flexible non-linear approach for modeling fish consumption showed a decrease in RA risk for one to three servings per week of fish consumption, followed by a slight increase in risk for higher consumption.et al., the inverse association was statistically significant only for broiled or baked fish and not for total fish consumption, as the presence of PCBs is lower in cooked fish [The inverse association between fish consumption and risk of developing RA observed in some of the studies has been mainly attributed to its content of long-chain n-3 polyunsaturated fatty acids (PUFAs) ,10,14. Iked fish .The main strength of this meta-analysis is its dose-response design, that provides better quantification of the associations between specified amounts of fish and risk of RA. A dose-response meta-analysis should be the preferred option when performing a systematic review, rather than running a meta-analysis based only on the comparison of the extreme categories of consumption (high versus low), which could vary considerably among studies.Among the limitations, a meta-analysis is deeply influenced by the quality of the single studies included. Results could be over- or underestimated due to residual confounding. Of the seven studies included in this meta-analysis, three ,12,13 adIn conclusion, the summary estimates from this dose-response meta-analysis show a non-statistically significant inverse association between fish consumption and RA.


The West-African (WA) Zaire Ebolavirus disease (EVD) outbreak was characterized by an exceptionally high number of cases and deaths as compared with the Eastern-Central African (ECA) outbreaks. Despite the Zaire Ebolavirus being the most lethal for humans, case-fatality rate, close to 80 % in ECA outbreaks, almost halved to 47 % in Guinea-Liberia-Sierra Leone (WA). Such an improvement was due to the remarkable implementation of international humanitarian aids. Some studies also suggested that the long human-to-human transmission cycle occurred in WA, gave rise to human adaptation and consequent immune escape. Haemorrhage, the main feature in seriously infected EVD patients, is due to the immune system that triggers the infected endothelial cells which expose the spike-like glycoprotein (GP) of the virion on their surface. If the human adaptation hypothesis holds true, the proportion of EVD patients with haemorrhage in the WA outbreak should be lower than in the ECA outbreaks due to immune escape. Therefore, the aim of this meta-analysis was to compare the relative frequencies of three typical haemorrhagic symptoms  in the ECA and WA outbreaks.Literature searches were performed through PubMed and Scopus using generic keywords; surveys including at least ten patients reporting CB, NB, GB relative frequencies were extracted and split into ECA and WA. The meta-analytical methods chosen were based on the levels of between-study heterogeneity and publication bias. Pooled CB, NB, GB relative frequencies in ECA and WA were estimated and compared. Subgroup analysis including only studies on Zaire Ebolavirus also was performed.Fifteen studies  were located with 4,867 (CB), 3,859 (NB), 4,278 (GB) EVD patients overall. GB pooled relative frequency was 45.3 %  and 18.0 % , in ECA and WA; NB was 10.6 %  and 1.3 % (1.0–1.8 %); GB was 24.2 %  and 1.9 % . Subgroup analysis confirmed these results.During the WA outbreak the relative frequency of GB decreased by two thirds, while NB and GB almost disappeared, suggesting that the Zaire Ebolavirus human adaptation hypothesis is plausible.The online version of this article (doi:10.1186/s12879-015-1302-4) contains supplementary material, which is available to authorized users. Megachiroptera) are naturally infected: Hypsignathus monstrosus (hammer-headed fruit bat), Epomops franqueti (singing fruit bat) and Myonycteris torquata (little collared fruit bat). Asymptomatically-infected bats drop partially eaten fruit and masticated fruit pulp, contaminated by their saliva and blood, to the ground, where they are eaten by great apes and forest duikers (antelopes). Ebolavirus may then be transmitted from these infected animals to humans. Humans and primates have been considered the end hosts and have not been regarded as reservoir species [Ebolavirus disease (EVD) is a zoonosis with persistence of the virus in reservoir species. Indeed, at least three species of fruit bats .Another element in support of this hypothesis is the change in case-fatality rate. Indeed, Zaire Ebolavirus is the most lethal species with an estimated case-fatality rate of 76 %, considerably higher than the Sudan (55 %) and the Bundibugyo (37 %) species . During Haemorrhage is a serious EVD consequence, often responsible for patients' demise. Actually, during the first EVD outbreak in South Sudan, almost all (91 %) of the fatal cases and only one half (48 %) of the nonfatal cases showed some visible bleeding features . This liTherefore, given the association between death and haemorrhage in EVD patients, in order to evaluate whether the large decrease in case-fatality rate observed during the West African outbreak was exclusively due to humanitarian aids or could be explained by Ebolavirus human adaptation, the current study sought to investigate the relative frequency of EVD patients with haemorrhagic features. More specifically, if the immune evasion hypothesis holds true, patients with haemorrhage should be less frequent during the West-African outbreak than during the Eastern-Central African outbreaks. If, alternatively, Ebolavirus adaptation to humans did not occur in West Africa, the frequency of EVD patients with haemorrhage did not vary. Thus, this meta-analysis sought to estimate the pooled relative frequencies of conjunctival and orofacial  haemorrhagic symptoms observed during the earlier Eastern-Central African and the latest West African outbreaks.A meta-analysis was performed to estimate the pooled relative frequency of the three main orofacial haemorrhagic symptoms/signs, that is, conjunctival bleeding/injection (and conjunctivitis), epistaxis and gingival bleeding, features chosen because they are easily detectable, although they are not exclusive of EVD. It was assumed that the other conditions and diseases associated with similar symptoms (e.g. gingivitis) did not change in frequency within the study populations throughout these years and, therefore, did not interfere with the present analysis.Literature searches were performed through Scopus and PubMed without time and language restrictions. Inclusion criteria were surveys reporting conjunctival, nasal and/or gingival bleeding in samples of at least ten EVD patients. Exclusion criteria were all non-relevant studies , case reports/samples of less than ten EVD patients, surveys which did not report bleeding features analytically.Generic key words were used to minimize selection bias which, in meta-analyses, refers to undetected published studies. Selection bias was considered more pervasive than publication bias, which refers to unpublished studies, due to the recent explosion of scientific journals and publications . Thus, kTitles and abstracts were used to exclude non-relevant studies. Full texts of remaining surveys were searched and read and only surveys which met the inclusion criteria were selected. The reviewers independently extracted conjunctival bleeding (EMV), epistaxis (LTM) and gingival bleeding (GAM) relative frequencies. Extracted data were then supervised and meta-analyzed by the two other authors (SP and CS).2 statistic [2 values the meta-analysis was performed with the fixed-effects method, for significant I2 values the random-effects method was used.The pooled relative frequencies of the three features were assessed. The meta-analytic method was chosen on the basis of the level of between-study heterogeneity, which was investigated with the Itatistic . For non0 method [Publication bias was formally investigated  with the0 method , 39.Sensitivity analysis to study inclusion was made to investigate whether the pooled relative frequency estimates were influenced by a single study.Surveys were then split according to the country where they have been performed into Eastern-Central African and West African outbreaks. Pooled relative frequency estimates of conjunctival nasal and gingival bleeding were assessed for these subgroups using the aforementioned methodology.Studies on conjunctival bleeding were split into studies which reported bleeding or injected eyes  and studies which reported more generic conjunctivitis. Pooled relative frequency estimates were assessed for all studies together, for Eastern-Central African studies and for West African studies.Subgroup analysis also was performed to investigate whether there were differences between Zaire Ebolavirus outbreaks. Therefore, since the West-African outbreak was exclusively due to the Zaire Ebolavirus species, surveys from Eastern-Central Africa reporting only Zaire Ebolavirus outbreaks were considered and the pooled relative frequency estimates of conjunctival nasal and gingival bleeding were assessed.This article followed the MOOSE guidelines for reporting meta-analyses of observational studies . The staThe Scopus and PubMed searches, performed on October 15, 2015, provided 130 and 691 studies, respectively , those considered for nasal bleeding were 3,859 , those considered for gingival bleeding were 4,278   and on the reporting protocol adopted by the healthcare staff. Although there are no elements to support this hypothesis, it is reasonable to conjecture that if the general population was unaware of EVD features, infected subjects with epistaxis, gingival bleeding and/or conjunctival bleeding as main features were less likely to present to healthcare facilities than were subjects more obviously ill with high fever, vomiting and/or diarrhoea. Few of the healthcare workers would be trained specifically in ENT/Ophthalmology and/or Dental examinations, and facilities for these were likely uncommon. Thus, primary studies must yield some degree of reporting bias, which probably led to underestimate the true relative frequencies of the these bleeding features. However, if reporting bias was present in the African healthcare facilities, it was probably lower in the studies used for this meta-analysis, because authors often followed the WHO EVD case definition recommendations, which comprised gingival bleeding and conjunctivitis among the key features .Another issue with the present meta-analysis was selection bias, which usually refers to published studies that have not been located. In this analysis it may also refer to the inclusion of partially duplicate studies, which reported data from the same sets of patients and to the exclusion of studies which were mistakenly considered duplicate and actually were original. This problem regards principally the West African outbreak, due to the exceptionally high number of published studies. In order to obtain the most reliable pooled estimates, the risk to exclude potentially original studies was preferred to the risk to reuse data from the same set of patients. Indeed, in the first case, the limited number of patients led to broader confidence intervals, while in the latter case, the inclusion of duplicate data may have raised the burden of the bias of the primary study, in the event that there was any.The present analysis suggests that conjunctival bleeding is the most frequent EVD orofacial symptom. However, it is not possible to diagnose EVD solely on the basis of conjunctival bleeding even if fever, vomiting and diarrhoea are co-existing, because these features are frequently present in many infectious diseases common in Eastern-Central and West Africa, such as measles  and malaSuch a huge decrease in ocular and orofacial EVD features, which occurred during the West African outbreak, parallels the significant decrease in case-fatality rate, which fell by one third, from 65 %  during the Eastern-Central African outbreaks , to 47.0Another element supporting these results showing that haemorrhagic features drastically decreased during the West African outbreak, is that in 20 % EVD patients the transmission route was unexplained . This suAn element of debate is the meaning of the doubled mutation rate of the Ebolavirus occurred during the West African outbreak. According to two studies performed on whole Zaire Ebolavirus genome and on GP glycoprotein sequence, there were no signs of positive selection, which would have definitively confirmed the human adaptation hypothesis , 19. ConThese data suggest that, although the above-mentioned social determinants have been responsible for part of the reduced case-fatality reported during the West-African EVD outbreak, human adaptation, along with immune evasion, may have occurred. Indeed, in the classification of stages leading to endemic human pathogens from animal pathogens, Ebolavirus was previously classified at stage 3, that is, an animal pathogen that can undergo only a few cycles of secondary transmission between humans, so that occasional human outbreaks triggered by a primary infection soon die out. However, the West African outbreak yielded the characteristics of a stage-4 pathogen, that is, an infectious disease that exists in animals and that infects humans cyclically by primary transmission from animal host, but that also undergoes long sequences of secondary transmission between humans, without the involvement of animal hosts. Examples of stage-4 infectious diseases are typhus, yellow fever and influenza A . AccordiIn line with this theory and accounting for the reported decreased proportion of EVD patients with haemorrhagic symptoms, it is possible to conjecture that the aforementioned West Africa-specific social determinants have been responsible for the initially high number of secondary (and undetected) transmissions between humans . Thanks In conclusion, along with the increased Ebolavirus mutation rate and the decreased case-fatality rate that occurred during the West African outbreak, this outbreak was also characterized by a significant decrease in the proportion of patients with bleeding features. Some of these features, that is, nasal and gingival bleeding almost disappeared. These data support the hypothesis of Ebolavirus adaptation to human host.


We performed a meta-analysis of all eligible studies assessing ITPA gene polymorphisms related to RBV-induced hemolytic anemia in HCV-infected patients published in PubMed, Embase and the Cochrane library prior to the end of 2014.There is growing evidence that variations in the gene encoding inosine triphosphate pyrophosphohydrolase (ITPase), known as Three outcomes were evaluated: (1) hemoglobin decline, (2) severe anemia, and (3) RBV dose reduction or treatment discontinuation. Pooled odds ratio (OR) and 95 % confidence interval (95 % CI) were estimated by either fixed or random effects models.ITPA rs1127354 CC genotype and absent ITPase deficiency haplotype were also associated with severe anemia {[OR = 7.77 ] and [OR = 4.79 ], respectively}. Additionally, the rs1127354 CC genotype showed significant association with RBV dose reduction or stopping treatment .Twenty-nine studies were selected from the literature search: 20 references involving 6533 individuals for hemoglobin decline, 13 references on 3764 patients for severe anemia, and 16 references on 3918 patients for RBV dose reduction or discontinuation. Significant associations with hemoglobin decline were found for rs1127354 CC [OR = 12.84 ], rs7270101 AA [OR = 3.41 ] and rs6051702 AA [OR = 4.43 ] genotypes. Moreover, significant associations with hemoglobin decline were also found for absent [OR = 6.01 ] and mild [OR = 4.68 ] ITPase deficiency haplotypes. The ITPA polymorphisms increase the likelihood of developing hemolytic anemia for HCV-infected patients on RBV-based therapy, particularly rs1127354 CC and rs7270101 AA genotypes, suggesting the utility of screening for ITPA polymorphisms to avoid hematological toxicity and increase adherence to RBV-based therapy.The online version of this article (doi:10.1186/s12967-015-0682-y) contains supplementary material, which is available to authorized users. For many years, pegylated interferon-alpha (pegIFNα) plus ribavirin (RBV) combination therapy has been the standard treatment for hepatitis C virus (HCV) infection, but the side-effects have made the therapy arduous for many patients . Recentlinosine triphosphatase (ITPA) gene, which encodes an inosine triphosphate pyrophosphohydrolase (ITPase), are associated with protection from hemolytic anemia during pegIFNα/RBV therapy . The sensitivity analysis showed that no study should be excluded  , but the pooled OR was not altered [OR = 2.70 ]. The sensitivity analysis showed that no study should be excluded   and the pooled OR was significant [OR = 4.43 ]   vs. mild (+)/moderate (++)/severe (+++) ITPase deficiency are shown in Fig. 2 = 5.7 %; p = 0.386)  vs. moderate (++)/severe (+++) ITPase deficiency are shown in Fig. 2 = 0.0 %; p = 0.819)  vs. severe (+++) ITPase deficiency are shown in Fig. ITPA polymorphisms and severe anemia .A total of 13 references were examined to evaluate the association between 2 = 32.7 %; p = 0.146)  , and the pooled OR decreased [OR = 2.80 ]. The sensitivity analysis showed that no study should be excluded  vs. mild (+)/moderate (++)/severe (+++) ITPase deficiency are shown in Fig.  95 % CI .74; 4.512 = 0.0 %; p = 0.874)  vs. moderate (++)/severe (+++) ITPase deficiency are shown in Fig. 74) Fig. c. The seITPA polymorphisms and RBV dose reduction or discontinuation   and the pooled OR was not altered [OR = 2.52 ]. Additionally, the sensitivity analysis showed that none of the 11 studies should be omitted from the analysis   vs. mild (+)/moderate (++)/severe (+++) ITPase deficiency are shown in Fig. 2 = 0.0 %; p = 0.489)  vs. moderate (++)/severe (+++) ITPase deficiency are shown in Fig. 2 = 0.0 %; p = 0.724)  vs. severe (+++) ITPase deficiency are shown in Fig. Several factors were analyzed to investigate the possible influence on the heterogeneity  was associated with a higher chance of developing hemoglobin decline. Additionally, significant associations with a higher chance of developing hemoglobin decline were found both absent (−) and mild (+) ITPase deficiency haplotypes. (2) The ITPA rs1127354 polymorphism and absent (−) ITPase deficiency haplotype were associated with severe anemia. (3) The rs1127354 CC genotype showed a significant association with RBV dose reduction or discontinuation of treatment.The three major results of our meta-analysis were: (1) The presence of the major alleles in homozygosis for Hemolytic anemia is an important side-effect in RBV-based HCV therapy –4. The tITPA genotypes  of the three SNPs studied  and RBV-induced hemolytic anemia. In most of the studies, the odds of developing anemia was more than double in patients with an unfavorable ITPA genotype than in patients with a protective ITPA minor variant. However, the magnitude of the association was different in many cases. This may be due to the different criteria considered in each individual study.In our meta-analysis, we found a significant association between the unfavorable ITPA polymorphisms have been associated with hemoglobin decline, few studies have compared the predictive value of combining the three ITPA polymorphisms, probably because of the scattered distribution of these ITPA polymorphisms among diverse ethnic groups and world populations. In our meta-analysis, a simultaneous evaluation of the three ITPA SNPs  was done when possible, but the rs6051702 polymorphism was rarely encountered and often there was too little data to draw robust conclusions on all three. The ITPA rs1127354 polymorphism (P32T substitution) exists at low frequency in Central and South American populations (1–2 %), at a constant frequency across Caucasian and African populations (6–7 %), and at the highest frequencies in Asian populations (14–19 %) [ITPA polymorphisms according to the study population.Although 14–19 %) . Also, t14–19 %) . For theITPA polymorphisms. There were two studies that showed the three SNPs at the same time [ITPA major alleles at the polymorphic sites rs1127354 and rs7270101, also showed a similar trend. Relationships in the same direction were found for severe anemia, but with fewer studies. However, almost no significant associations were found for ITPA polymorphisms or haplotypes with RBV dose reduction or discontinuation. This might be explained by the multiple factors influencing this outcome or due to the inclusion of different events in the same endpoint (RBV dose reduction and/or discontinuation), which are reported at different times during treatment. In addition, the need for therapy modification may be influenced by other clinical factors that were not taken into account. Of special interest is the use of erythropoietin (EPO) to improve hemoglobin levels and limit the need to reduce the dose of RBV [The decrease in hemoglobin levels was the outcome most frequently studied and the only one that provided enough data to compare the three ame time , 29, ande of RBV . To datee of RBV , 33, 35,The heterogeneity of the studies must be taken into account in a meta-analysis. The covariates from the study population that were included in the analysis were age, gender and HIV coinfection, which have been previously described as risk factors for RBV-induced anemia , 42. AnoITPA SNP and anemia has been reported in patients on RBV monotherapy [ITPA rs1127354 is a useful predictor of the development of severe anemia in telaprevir-based triple therapy patients [ITPA polymorphism was not associated with early anemia or therapy modification [The severity of RBV-related anemia is known for long but it is difficult to assess the role of each drug  in the anemia. Although most of the studies have been performed in patients on combined therapy (pegIFNα/RBV), a strong association between an otherapy . Moreoveotherapy . Data frotherapy –36, withpatients , 36, AghITPA polymorphisms and the second-generation of DAAs, such as sofosbuvir, simeprevir, and daclatasvir. Note that patients undergoing triple therapy with pegIFN/RBV plus these new DAAs have similar anemia frequencies and profiles as patients receiving pegIFN/RBV alone [ITPA polymorphisms will still be useful in preventing anemia while RBV continues to be included in HCV treatment regimens.To date, no data have been available about BV alone , 51. In BV alone . Thus, IFinally, in order to properly interpret our results, some considerations have to be taken into account. Firstly, our meta-analysis was performed by using the unadjusted raw data provided from each study, whereas most of the results given by the authors had been adjusted by age, gender, HCV viral load, and/or other factors. For this reason, the pooled ORs may differ slightly from those reported in the original articles. Secondly, renal function, baseline hemoglobin level and other variables involved in determining individual susceptibility to RBV-induced anemia have not been considered. Thirdly, most studies reported the results as haplotype, which does not allow the results of each polymorphism to be evaluated separately. Fourthly, the majority of the studies had a retrospective design and the number of studies in some subgroup analyses was small, which might have led to weak results. Consequently, these results should be interpreted with caution.ITPA polymorphisms increase the likelihood of developing hemolytic anemia in HCV-infected patients on RBV-based therapy, rs1127354 CC and rs7270101 AA genotypes, suggesting the utility of screening for ITPA polymorphisms as a way to avoid hematological toxicity and increase adherence to RBV-based therapy.In conclusion,


Flavonoids have been suggested to play a chemopreventive role in carcinogenesis. However, the epidemiologic studies assessing dietary intake of flavonoids and esophageal cancer risk have yielded inconsistent results. This study was designed to examine the association between flavonoids, each flavonoid subclass, and the risk of esophageal cancer with a meta-analysis approach. We searched for all relevant studies with a prospective cohort or case-control study design published from January 1990 to April 2016, using PUBMED, EMBASE, and Web of Science. Pooled odds ratios (ORs) were calculated using fixed or random-effect models. In total, seven articles including 2629 cases and 481,193 non-cases were selected for the meta-analysis. Comparing the highest-intake patients with the lowest-intake patients for total flavonoids and for each flavonoid subclass, we found that anthocyanidins , flavanones , and flavones  were inversely associated with the risk of esophageal cancer. However, total flavonoids showed marginal association with esophageal cancer risk . In conclusion, our study suggested that dietary intake of total flavonoids, anthocyanidins, flavanones, and flavones might reduce the risk of esophageal cancer. Esophageal cancer ranks as the 10th most common malignancy and the eighth most common cause of cancer-related mortality worldwide. An estimated 455,800 newly diagnosed esophageal cancer cases and a related 400,200 deaths occurred worldwide in 2012 ,4,5. EpiHigh intake of fruits and vegetables is associated with beneficial health effects , and thiMost of the cancer preventive effects of flavonoids have been shown in animal and cell culture studies ,18,19. FA comprehensive literature search was conducted to assess the associations between total flavonoids, flavonoid subclasses, and esophageal cancer. Published papers were identified from three electronic databases: PUBMED , EMBASE The following inclusion criteria were used for the present meta-analysis: (1) original articles about the association between total flavonoids, flavonoid subclasses, and esophageal cancer; (2) the study design is cohort or case-control study; (3) total flavonoids or flavonoid subclasses intake was estimated by food frequency questionnaire (FFQ) and the flavonoid food composition database; (4) relative risk (RR), hazard ratio (HR), or odds ratio (OR), and corresponding 95% confidence intervals (95% CI) were available; (5) published in English.Accordingly, the following exclusion criteria were also considered: (1) cell studies and animal studies; (2) abstracts, reviews, letters to the editor, case reports, and repeated publications; (3) data related to exposure assessment (blood/urinary levels) of total flavonoids or one of the flavonoid subclasses; (4) studies without sufficient data for estimating the OR with 95%CI; (5) published in other languages.Originally, we included RCTs in our search criteria, but because there were no RCTs of flavonoid intervention, no RCTs were included in the present study.versus the lowest flavonoid exposure and their 95% CI; (8) confounders adjusted for in multivariate analysis.According to the Meta-Analysis of Observational Studies in Epidemiology guidelines , two rev2I statistics of 25%, 50%, and 75% indicate low, moderate, and high levels of heterogeneity, respectively. If p < 0.05 and I2 > 50%, a random-effect model based on the DerSimonian and Laird method was used to calculate pooled ORs (95% CIs). Otherwise, a fixed-effect model based on the Mantel–Haenszel method was used [Six case-control studies provided ORs and one cohort study provided HR together with their corresponding 95% CIs. Since the absolute risk of esophageal cancer was low, the OR was theoretically similar to HR . Therefowas used . Meta-rep < 0.10) [p values were for a two-sided test, and p < 0.05 was considered statistically significant.Potential publication bias was examined by funnel plot and Egger’s test ( < 0.10) . The senet al. only investigated the effect of three phytochemicals, including resveratrol, quercetin, and lignans on esophageal cancer [et al. [. All of the individual studies were adjusted for a wide range of potential confounders, including age, sex, race, education, energy intake, BMI, physical activity, parity, smoking, and alcohol drinking.With our search strategy, we identified 149 citations, 466 citations, and 144 citations from PUBMED, EMBASE, and Web of science, respectively. After removing 292 duplicate papers, 577 articles remained. Twelve articles were reviewed in full text after reviewing the titles and abstracts. Among them, two articles reported results for a duplicate population ,42, one l cancer . Because [et al. . All of vs. lowest categories of total flavonoids and each flavonoid subclass, respectively. As shown in I2 = 61.0, p = 0.012), flavanones , and isoflavones . However no substantial heterogeneity existed across the studies of anthocyanidins , flavan-3-ols , flavones  and flavonols  and proanthocyanidins . Overall, the risk of esophageal cancer significantly decreased in patients with the highest intake of anthocyanidins , flavanones , flavones  by 40%, 35%, and 22%, respectively, compared to patients with the lowest intake of total flavonoids, anthocyanidins, flavanones, and flavones. However, marginal association of total flavonoids  and no significant association of flavan-3-ols , flavonols , isoflavones , or proanthocyanidins  with esophageal cancer risk was observed.We identified eight studies of total flavonoid intake and esophageal cancer, seven studies of anthocyanidins, flavan-3-ols, flavanones, flavones, flavonols and isoflavones, four studies of proanthocyanidins and one study of lignans, quercetin, and resveratrol. We calculated the pooled ORs of esophageal cancer risk for the highest p = 0.043) was the source of heterogeneity, source of control (p = 0.072) was a possible source of heterogeneity, and study design, pathological type (p = 0.860), geographic location (p = 0.368), period of dietary assessment (p = 0.850), and adjustment for energy (p = 0.850) showed no significant impact on between-study heterogeneity. When stratified by pathological type, source of control, geographic location, dietary assessment, length of dietary recall, and adjustment for energy intake, no statistically significant heterogeneity was found in the studies of EAC , studies with population-based control population , studies conducted in European populations , studies using validated FFQ for flavonoid intake estimation , studies in which dietary intake was recalled at least five years before diagnosis , and studies without adjustment for energy intake .Substantial heterogeneity existed across studies of total flavonoid intake, and meta-regression and subgroup analysis were performed to find the source of heterogeneity. Meta-regression analysis found dietary assessment (p = 0.454). Funnel plots were provided in Sensitivity analysis suggested that no individual study significantly affected the pooled OR, which indicated that our results were statistically robust . Egger’sFlavonoids are a large group of ubiquitous polyphenolic secondary metabolites in plants with a wide range of properties, including a widely reported anti-cancer effect. It is hypothesized that flavonoids can reduce the risk of cancer by reducing oxidative damage , quenchiTo date, the association between dietary flavonoid intake and various cancer risks has remained inconclusive. The published meta-analyses have found that total dietary flavonoid intake was not associated with a reduced risk of colorectal or stomach cancer, and dietary intake of flavonols, flavan-3-ols, anthocyanidins, and proanthocyanidins showed a significant inverse association with colorectal cancer risk. However, a significant association was found only between flavonols and stomach cancer risk based on cohort studies . The latIn vitro, flavones  were able to induce cytotoxicity in KYSE-510 cells in a dose- and time-dependent manner. The effect of flavanones on esophageal cancer has been only scarcely investigated previously. Therefore, more research on the mechanism of flavones and flavanones on the development of esophageal cancer should be carried on in future.The present meta-analysis indicated that anthocyanidins intake was significantly reduced the risk of esophageal cancer. These compounds provide pigmentation to many fruits and vegetables, such as berries, red grapes, purple sweet potato, and red cabbages . As blacGiven that heterogeneity existed across studies of the total flavonoid intake, meta-regression and subgroup analysis was performed to find the source of heterogeneity. Meta-regression analysis found dietary assessment was the source of heterogeneity, source of control was a possible source of heterogeneity. When stratified by pathological type, source of control, geographic location, dietary assessment, length of dietary recall, and adjustment for energy intake, the heterogeneity decreased; no significant heterogeneity was found in the studies of EAC, studies with population-based controls, studies conducted in European populations, studies using validated FFQ for flavonoids intake estimation, studies in which dietary intake were recalled for at least five years before diagnosis, and studies without adjustment for energy intake.In our view, the present meta-analysis has several advantages. First, this study is the first comprehensive meta-analysis evaluating the potential association between dietary intake of flavonoids and esophageal cancer risk. Second, all of the individual studies were adjusted for a wide range of potential confounders, including age, sex, race, education, energy intake, BMI, physical activity, parity, smoking, and alcohol drinking. Third, in addition to the estimation between total flavonoids and esophageal cancer risk, the association between subclasses of flavonoids and esophageal cancer risk was also assessed. However, several limitations should be also taken into consideration. First, the number of studies involved in the meta-analysis was not large enough. Second, this meta-analysis had regional restrictions, as all of the included studies were conducted in western countries. Third, most of the eligible studies were case–control studies, in which the information recorded about past dietary assessment would have been prone to recall bias. Selection bias often occurs if the recruited cases or controls are systematically different from the population of people who they are intended to represent in case-control studies. Fourth, the included cohort study conducted in 10 Europe countries used country specific dietary questionnaires, which might influence the results. Finally, we did not conduct the dose-response analysis of the relationships for the intake of flavonoids and esophageal risk for the irregularity and incomplete data of flavonoid intake.In conclusion, the present study indicated that dietary intake of total flavonoids, anthocyanidins, flavanones, and flavones—but not intake of other flavonoid subclasses—was associated with a decreased risk of esophageal cancer. Therefore, more carefully designed studies, especially cohort studies or randomized controlled trials, should be conducted to investigate the association of dietary flavonoids intake with the risk of esophageal cancer worldwide.


CST3, previously confirmed by meta-analysis to be associated with AD, is associated with exudative AMD. To our knowledge, this is the first report highlighting a genetic variant that increases the risk of developing both AD and AMD. Furthermore, we demonstrate that the risk associated with the mutant allele follows a recessive model for both diseases. We perform an AMD-CST3 case–control study genotyping 350 exudative AMD Caucasian individuals. Bringing together our data with the previously reported AMD-CST3 association study, the evidence of a recessive effect on AMD risk is strengthened . This effect closely resembles the AD-CST3 recessive effect  previously established by meta-analysis. This resemblance is substantiated by the high correlation between CST3 genotype and effect size across the two diseases (R2 = 0.978). A recessive effect is in line with the known function of cystatin C, a potent enzyme inhibitor. Its potency means that, in heterozygous individuals, a single functional allele is sufficient to maintain its inhibitory function; only homozygous individuals will lack this form of proteolytic regulation. Our findings support the hypothesis that recessively acting variants account for some of the missing heritability of multifactorial diseases. Replacement therapy represents a translational opportunity for individuals homozygous for the mutant allele.Age-related macular degeneration (AMD) and Alzheimer’s disease (AD) are degenerative, multifactorial diseases involving age-related accumulation of extracellular deposits linked to dysregulation of protein homeostasis. Here, we strengthen the evidence that an nsSNP (p.Ala25Thr) in the cysteine proteinase inhibitor cystatin C gene The online version of this article (doi:10.1007/s00439-015-1552-7) contains supplementary material, which is available to authorized users. APOE gene is associated with both diseases but quite intriguingly has opposing directions of effect. Whereas the APOE ε4 allele increases risk of developing AD, it decreases the risk of AMD  and Alzheimer’s disease (AD) are progressive neurodegenerative diseases exhibiting some common characteristics. A physical characteristic of both diseases is the presence of insoluble deposits at the site of pathogenesis. These pathological deposits—the amyloid plaques of AD and the drusen of AMD—demonstrate some compositional similarity, engender a pro-inflammatory response and impair essential cellular functions such as trafficking and secretion. These similarities indicate that common/similar cellular mechanisms may contribute towards the pathogenesis of both diseases. Certain environmental risk factors, such as smoking and obesity, are known to increase the risk of both diseases, along with age which is the major risk factor for both conditions. With respect to genetic risk factors the CST3) has also been implicated as a risk factor for both AD  in the signal sequence (p.Ala25Thr due to a c.G73A substitution) which results in an alternate homologue referred to as variant B. Cystatin C is a potent inhibitor of cysteine proteases and multiple lines of evidence (from molecular studies) support the hypothesis that wild-type cystatin C has a protective role against both these age-related diseases , while heterozygous individuals were not at significantly increased risk , indicating that the risk allele acts recessively . The genetic association between CST3 and AMD has been less well studied, with only a single case–control study reported to date, in which an association between exudative AMD and the polymorphism was highlighted , whereas heterozygotes were not at significant risk . This identical recessive effect of CST3 on AMD and AD risk is intriguing. In the line of the above, the main aim of this study was to further investigate the AMD-CST3 association.Meta-analysis of 8 association studies has confirmed that this SNP is associated with AD in Caucasians  were recruited (age range 65–96 with mean 80.1 years). Written informed consent for all participants used in this study was obtained for research use and approved by the Leeds (East) Research Ethics Committee. The diagnosis of exudative AMD was provided by ophthalmologists based on baseline stereoscopic colour fundus, fluorescein and indocyanine green angiogram images to identify lesion characteristics  (January 2014). This provided genotype information for 3781 Caucasians from the USA, which are assumed to contain undiagnosed AMD cases with a frequency equivalent to the Caucasian prevalence. We inferred that 2442 (64.5 %) of this sample are male, in that they have genotype information for the SRY gene.Population controls were taken from the largest publicly available online database Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA (http://frodo.wi.mit.edu/). Polymerase chain reaction (PCR) generated a 1292-bp product using forward primer CST3LRIIF 5′-CAGGAGTGGAGGAGGGAGATG-3′ and reverse primer CST3LRIIR 5′-CCAGATGAGGGGCTCTGTTTT-3′. This product contains three SNPs  in strong linkage disequilibrium, such that the genetic variation can be explained by two haplotypes, known as variant A and variant B. Two of the SNPs are located in the 5′ untranslated region and the third is located in exon 1 (leading to the missense p.A25T). Briefly, the PCR consisted of 40 ng of genomic DNA, 2pM of each forward and reverse primer, 1M Betaine and HotShot Mastermix . An initial denaturation step of 95 °C for 12 min was followed by 40 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 60 s. A final extension of 75 °C for 5 min completed the reaction. PCR products were electrophoresed on a 1.5 % agarose gel stained with ethidium bromide after which the gel was visualized using the ultraviolet light filter on the ChemiDoc Imaging system (BioRad).Genomic DNA was extracted from peripheral blood leucocytes by standard methods. Primers were designed using the online software Primer3 v.0.4.0  and sequencing reactions were carried out using Big Dye Terminator Cycle Sequencing V3.1 Ready Reaction Kit . To determine the sequence at SNPs rs5030707, rs73318135 and rs1064039 nested reverse CST3LRR primer 5′-GGCTCCTGGAAGCTGATCTTAG-3′ was used. To confirm the sequence a second nested reverse primer CST3BIIR 5′-TTGCTGGCTTTGTTGTACTCGC-3′ was used. The sequence data obtained from both primers was compared to see if they matched and together these data were used to determine the haplotypes. The sequencing reactions were run on an ABI3130xl Genetic Analyser and the data analysed for respective SNPs using Sequence Analysis 5.2 software (Applied Biosystems). Representative chromatograms of each of the three genotypes are presented in Supplementary Fig. S2.β). By calculating these parameters for both the heterozygote (βAG) and homozygote (βAA), we are able to make inferences about the genetic model of inheritance. Explicitly we test for the recessive model by testing the null hypothesis H0: d = βAA − βAG = 0, Ha: d > 0 as previously described . From this simulated case–control dataset we perform a two-tailed z test  at α = 0.05. After 10,000 iterations the number that successfully detected an association is used to estimate power. This is repeated for our study by changing the sample sizes of cases and controls accordingly. For the two-study meta-analysis, power calculation requires simulating the two case–control data sets for each iteration and performing the z test based on the weighted normal distribution .To determine the power of the association study of Zurdel et al.  a singleCST3 SNP (rs1064039) and AD has been established by meta-analysis , whereas the heterozygote risk is non-significant for both diseases . Thus a recessive model of inheritance best explains the association with CST3 for both diseases. To support this recessive model we confirm that homozygote effect size is significantly greater than the heterozygote effect size in both AD (P = 0.010) and in AMD (P = 0.013). Put together the risk “A” allele is recessive for both diseases; only individuals with two copies of it are at a significantly higher risk of developing AD and AMD.Association between the T3 SNP rs064039 anAA for AD and AMD, we also calculated how probable it would be to simultaneously observe both these ORs by chance given the null hypothesis that CST3 has no effect on both diseases. We find that such a set of observations is very unlikely to happen by chance (P = 5.0 × 10−4). To further quantify the similarity between the CST3 genotype data of the two diseases, we calculate the coefficient of determination of the four variables  as a reasonable estimate for its AMD effect size. Using this assumption and a z test for recessive effect we calculate the power of Zurdel’s study  to be 24.6 %. Thus for every four studies of such size, only one would detect the association.To estimate the power of an association study an estimate of the effect size of the alternative hypothesis is required. Because both molecular and epidemiological evidence support homogeneity between AMD and AD with respect to A, would be 1.15 given a true recessive effect of ORAA = 1.73 . From further study of this relationship we found that for a given recessive effect size, ORNN, the perceived per-allele ORN is linearly related to the allele frequency of the SNP  and moderate allele frequency (10 %) can appear to have a weak effect from its per-allele OR (ORN = 1.2).We also estimated the power of a GWAS to detect an association with a variant with this recessive effect size. Using the sample sizes, test and significance level of an existing AD GWAS  was genotyped in Caucasian AMD patients from England (n = 350). We tested this AMD data against the Exome Sequencing Project control data as it was the largest publically available set of population controls (n = 3781). In this control sample the frequency of the variant allele “A” is 17.5 % and the proportion with the “AA” genotype is 3.0 %. Thus the data are in Hardy–Weinberg equilibrium (P = 0.76) and also fall within the allele frequency range reported from the Caucasian studies in the AD meta-analysis, which ranged from 17.1 to 22.8 %.On observing these findings we sought to replicate the finding of Zurdel et al. in investigating the association between P = 0.25). Although not significant, it is the “AA” homozygotes that are at greatest risk  compared to the heterozygotes , with “GG” homozygotes as baseline. Thus our data are consistent with the recessive effect observed previously in both AMD and AD, but are not powerful enough to reach significance by itself. Further indication of an effect was obtained by performing the analysis only on AMD cases aged above 80 years . However, all further analyses in this study are performed on the total AMD dataset .Case–control analysis of these results exhibits a highly similar pattern of genotype risks to those observed by Zurdel Table , but is AA = 1.73), we calculate it would require a sample of 735 AMD cases, whilst maintaining the control sample size of 3781.We calculate the power of our study alone to be 53.7 %, meaning that around half of studies this size would fail to detect the association given the effect size reported for AD. The relatively low powers presented so far are likely due to the frequency of the homozygote risk genotype. For instance within our sample of 350 AMD cases, only 16 (4.6 %) are “AA” homozygotes  . We also repeated the random effects meta-analysis using a meta-regression approach  with the conventional random effects meta-analysis.We proceeded to perform a preliminary meta-analysis to bring together the results of the two AA under the null hypothesis of CST3 having no effect on either disease. We found that this updated set of observations was even more unlikely to happen by chance (P = 7.8 × 10−5) than previously calculated. We then applied an AMD meta-analysis to the “AG” heterozygotes versus the baseline “GG” genotype . Finally, we compared the AMD and AD effect sizes estimated from their respective meta-analysis alongside one another and observed a striking similarity , supporting the hypothesis that homogeneity exists between AMD and AD risk with respect to CST3 genotype.We calculated how probable it would be to simultaneously observe both ORity Fig. . Using tCST3 associated with both diseases but there is a striking similarity in the underlying model of inheritance, namely a recessive genetic model. We first noticed this similarity by bringing together an AD-CST3 meta-analysis and the only reported association study between CST3 and AMD. Under the null hypothesis that both diseases are not affected by CST3 genotype the combined observed data are very unlikely to occur by chance (P = 5.0 × 10−4). However, we estimated the power of this AMD association analysis to be fairly low (24.6 %), assuming the AMD effect size is equivalent to AD. On repeating the AMD association study, again the same recessive trend was observed with only the homozygote variants at elevated risk. Taken together a meta-analysis of the two AMD-CST3 studies finds a significant association (P = 0.005) with an increased estimated power of 67.7 %. The recessive trend is strikingly similar between the two diseases  given the null hypothesis that CST3 has no effect on both diseases.We bring together AD and AMD case–control data and observe that not only is CST3 in AMD pathogenesis. It is also important to note that both of these AMD association studies were performed with Caucasian samples only. With AD the association with CST3 was only found in Caucasian samples, while in Asian samples no significant AD-CST3 association was detected  is concealed when only considering its additive or per-allele effect size (ORA = 1.15). Herein, we propose that this explanation also serves as a hypothesis to account for some of the current “missing heritability” for common diseases. For AMD only 15–65 % of total heritability is explained by the 19 loci detected so far  can be much stronger  between an SNP 1.3 kb downstream of CST3 (rs6048952) and plasma levels of cystatin C   Fig. . Decreas99) Fig. .Fig. 5PaCST3 is implicated in AMD pathogenesis. In particular, only individuals homozygous for the variant allele are at increased risk. Intriguingly the same recessive effect is observed at the same SNP with AD risk. This finding corresponds with previous evidence from both AD and AMD in vitro models. Observing a recessive effect implies that a single wild-type allele is able to compensate for the mutant allele. This may be due to cystatin C being a potent inhibitor of cysteine proteases (inhibitory constant ki for cathepsin B is 0.25 nM)