CHEK2 confer increased risk of breast cancer. However, no large-scale study has used full open reading frame mutation screening to assess the contribution of rare missense substitutions in CHEK2 to breast cancer risk. This absence has been due in part to a lack of validated statistical methods for summarizing risk attributable to large numbers of individually rare missense substitutions.Both protein-truncating variants and some missense substitutions in in silico assessment of missense substitutions used for analysis of unclassified missense substitutions in BRCA1 and BRCA2 to the problem of assessing candidate genes using rare missense substitution data observed in case-control mutation-screening studies. The method involves stratifying rare missense substitutions observed in cases and/or controls into a series of grades ordered a priori from least to most likely to be evolutionarily deleterious, followed by a logistic regression test for trends to compare the frequency distributions of the graded missense substitutions in cases versus controls. Here we used this approach to analyze CHEK2 mutation-screening data from a population-based series of 1,303 female breast cancer patients and 1,109 unaffected female controls.Previously, we adapted an CHEK2 missense substitutions. Additional findings were that (1) the risk estimate for the most severe grade of CHEK2 missense substitutions (denoted C65) is approximately equivalent to that of CHEK2 protein-truncating variants; (2) the population attributable fraction and the familial relative risk explained by the pool of rare missense substitutions were similar to those explained by the pool of protein-truncating variants; and (3) post hoc power calculations implied that scaling up case-control mutation screening to examine entire biochemical pathways would require roughly 2,000 cases and controls to achieve acceptable statistical power.We found evidence of risk associated with rare, evolutionarily unlikely CHEK2 harbors many rare sequence variants that confer increased risk of breast cancer and that a substantial proportion of these are missense substitutions. The study validates our analytic approach to rare missense substitutions and provides a method to combine data from protein-truncating variants and rare missense substitutions into a one degree of freedom per gene test.This study shows that  BRCA1, BRCA2, PTEN and TP53 (Mendelian Inheritance in Man numbers (MIMs) 113705, 600185, 601728 and 191170, respectively); rare intermediate-risk variants, such as protein-truncating mutations in ATM [BRIP1 [CHEK2 [PALB2 [FGFR2, TOX3 (TNRC9), MAP3K1 and LSP1 [BRCA1, BRCA2, TP53 and PTEN account for approximately 20% to 25% of the familial risk of breast cancer, and adding the known intermediate-risk genes increases the proportion by perhaps 1% for each gene [Familial clustering of breast cancer is well recognized, having been described over 140 years ago ,3. Three2 [PALB2 ,11  and rare missense substitutions (rMS) [CHEK2 and then extrapolate the results to determine the requirements for much larger-scale studies.During the 1990s, linkage analysis proved to be an effective genome-wide approach for finding high-risk susceptibility genes for breast and colon cancer. Over the past few years, GWASs have proved to be an effective genome-wide approach to finding common, not necessarily causal, SNPs associated with modest risk. Case-control mutation screening, or its quantitative trait homolog of comparative mutation screening of individuals from the opposite ends of a trait spectrum, is emerging as a useful strategy for identifying and characterizing intermediate-risk susceptibility genes ,10,27-29CHEK2 mutation-screening studies and analyses described here were approved by the institutional review board (IRB) of the International Agency for Research on Cancer, the University of Utah IRB and the local IRBs of the Breast Cancer Family Registry (Breast CFR) centers from which we received samples. All participants gave written, informed consent.The Patients were selected from among women gathered by population-based sampling by the Breast CFRs at three centers  and the University of Melbourne) . PatientN = 1,313) were diagnosis at or before age 45 years and self-reported race or ethnicity plus grandparents' country of origin consistent with Caucasian, East Asian, Hispanic/Latino or African American racial or ethnic heritage.Selection criteria for cases  were frequency matched to cases within each center on racial or ethnic group, with age at selection not more than ± 10 years difference the age range at diagnosis of the patients gathered from the same center. Because of the shortage of available controls in some ethnic and age groups, the frequency matching was not one-to-one in all subgroups.The controls (CHEK2 amplicons harboring SNPs with a frequency ≥1% in either the Single Nucleotide Polymorphism Database (dbSNP) [ATM [CHEK2 exons 10-14 (which are involved in a subtelomeric repeat) relied on a long-range PCR assay as described by Sodha et al. [Mutation screening started from whole-genome amplified (WGA) DNA for coding exons 1-9 and from genomic DNA for exons 10-14. A nested polymerase chain reaction (PCR) strategy was used, followed by high-resolution melting (HRM) curve analysis ,32 and t (dbSNP)  or initi (dbSNP) . The labNP) [ATM , except a et al. .16NYAG^ (acceptor) (where ^ indicates the position of the splice junction), were reamplified from genomic DNA for confirmation of the presence of the variant. Because of the presence of pseudogenes that partially matched the sequence of the CHEK2 long-range PCR exons (exons 10-14), sequence variants identified within these exons were subsequently tested using allele-specific PCR assays for the primary PCR to confirm that the sequence variants initially identified were true CHEK2 variants. To ensure amplification of the CHEK2 DNA sequence and not amplification of the potentially interfering CHEK2 pseudogenes, the positions of the specific primers were chosen so that the 3' extremity bases perfectly matched the CHEK2 wild-type sequence, while they mismatched the corresponding position of the pseudogenes.All exonic sequence variants, plus splice junction consensus sequence variants that reduced splice junction sequence similarity to the standard consensus sequences AG^GTRRGT (donor) or YCHEK2 coding sequence were excluded from further analyses (n = 24). Process and data management of the mutation screening were carried out as described by Voegele et al. [All samples that failed at the primary PCR, secondary PCR or sequencing reaction stage were reamplified from WGA DNAs or genomic DNA. Samples that still did not provide satisfactory mutation-screening results for at least 80% of the e et al. . Primer Strongylocentrotus purpuratus) to analyze a small number of CHEK2 missense substitutions and in-frame deletions [Tetraodon nigroviridis) sequence with a full-length zebrafish (Danio rerio) sequence and including predicted CHK2 sequences from elephant (Loxodonta africana), platypus (Ornithorhynchus anatinus), tunicate  and fruit fly (Drosophila melanogaster). The alignment was characterized by (1) determining percentage sequence identity between each pair of sequences in the alignment, (2) using the Protpars routine of Phylogeny Inference Package version 3.2 software  software suite of alignment tools ,38 to preletions . We updasenstein ) to makesenstein ,42. The senstein . Missensign-GVGD -45 and Sign-GVGD ,42 softwign-GVGD ,47.To assess risk associations using the case-control frequency distribution of T+SJVs and rMSs, we constructed a single table with one entry per participant; zero or one rare sequence variant per participant; and annotations for type of sequence variants, study center, case-control status, race or ethnicity, and age. For the two participants who carried more than one rare variant of interest (one participant carried p.I448S (C15) plus p.E394D (C35), and one participant carried p.E239K (C15) plus p.R346H (C25)), only the variant belonging to the more likely evolutionarily deleterious grade  was considered.Most analyses were performed using multivariable unconditional logistic regression using Stata version 11 software . Differences in the case-control ratio between ethnic groups and age categories were accounted for by including categorical variables for each age category and ethnic group. Adjustment was also made for study center. We explored the possibility of interactions between ethnic group and study center, checking both improvement of model fit by the likelihood ratio statistic and comparing the estimates of the parameter of interest (log odds ratio (OR) per Align-GVGD grade) in different models. Adjustment for ethnic group should also capture confounding of genetic and social factors with interaction terms, allowing that this confounding effect may be different for the broadly labeled ethnic groups in different centers. Because the Breast CFR matched cases and controls for age in 5-year categories, and because the maximum age of Breast CFR patients included in this study was 45 years, all participants ages 41 years and older (at diagnosis for patients and at ascertainment for controls) were combined into a single age category.P values (Ptrend) were estimated from the resulting lognormal ORs using the logit function of Stata software. Carriers of T+SJVs were analyzed against the same noncarrier group defined above. Two strategies were used to combine evidence of association with T+SJV and rMS variants: (1) carriers of T+SJVs were combined with carriers of C65 rMSs in category 7, and (2) T+SJV carriers were assigned row label 8. We used the Fisher's exact test to obtain the lower bound of the 95% confidence interval (95% CI) for associations with categories that contained one or more patients but zero controls.Logistic regression trend tests were formatted such that participants who did not carry any T+SJV or any rMS, as well as carriers of the seven grades of rMSs  defined by Align-GVGD [Post hoc power calculations were performed by specifying a hypothetical OR and population prevalence for each class of variant, together with the cumulative probability of breast cancer prior to age 70 years. The ORs and control carrier frequencies that we specified for the individual grades of sequence variants, relative to the noncarriers, were based on data from the population-based Breast CFR sample series. For the grades for which there were a reasonable number of observations, that is, C0, C15, C25, C65 and T+SJV, we used the adjusted ORs and observed carrier frequencies. Because of the very low numbers of observations in grades C35-C55, ORs for these categories were estimated from the logistic regression OR coefficient and population carrier frequencies defined to obtain the specified OR, given the number of observations in patients. On the basis of these OR and frequency estimates, we calculated expected values and variances of the test statistics for the types of test considered: Pearson's χ2 test for the two-category tests and the Wald statistic from a logistic regression for the trend test. We then calculated the probability of these statistics exceeding a series of desired P value thresholds using a normal approximation.post hoc power calculations.Attributable fractions were estimated according to the method described by Greenland , and famCHEK2 mutation-screening amplicons was greater than 20%  were excluded because their PCR failure rate for CHEK2 revealed three distinct nonsense substitutions and four distinct small insertion deletion variants that should result in a truncated protein. One of these, c.1100delC, a well-known Northern European founder mutation that has been shown beyond any reasonable doubt to confer a moderately increased risk of breast cancer [P = 0.005) , but since none of this set of controls were found to carry such a variant, we could not estimate the OR.Full open reading frame mutation screening of t cancer , was obs5) Table . Howeveris study ,51, we nCHEK2 missense substitutions .In the course of this mutation screening, we observed 34 distinct Strongylocentrotus purpuratus) sequence  (Table CHEK2 variant). The OR for C65 rMSs was 8.75 (P = 0.044) (Table Ptrend = 0.0055) (Table P = 0.18) and were omitted. While removing the study center did not significantly reduce the goodness of fit (P = 0.12), this adjustment was retained on the grounds of prior plausibility.Using this alignment, we scored the 34 missense substitutions with Align-GVGD -45 and SCHEK2 protein multiple sequence alignment having an average of at least three substitutions per position and in which the farthest diverged sequence was from the (deuterostomate) sea urchin (Strongylocentrotus purpuratus). Our analysis thus conformed to the conditions under which Align-GVGD was calibrated and was used to grade missense substitutions in ATM [CHEK2 alignment in which the farthest diverged sequence was from the (protostomate) fruit fly (Drosophila melanogaster), which reached SIFT's median sequence conservation score threshold of 3.25, the OR for the SIFT score 0.00 grade was 3.03 and the logistic regression trend test gave Ptrend = 0.012 (Table Ptrend = 0.014 (data not shown). PolyPhen-2 uses a combination of its own precompiled protein multiple sequence alignments and crystal structure information to score missense substitutions. Using PolyPhen-2, we also set up three rMS grades: (1) the program's standard "Benign" grade, (2) its standard "Possibly Damaging" grade, and (3) its standard "Probably Damaging" grade. The OR for the Probably Damaging grade was 3.87, and the logistic regression trend test gave Ptrend = 0.0070. The rMS grades obtained with SIFT and PolyPhen-2 are also included in Supplementary Table S1 in Additional file We emphasize that our preplanned rMS analysis was based on rMS grading using Align-GVGD with a s in ATM ,45. In as in ATM ,42 and Ps in ATM ,47. WithCHEK2 gene model . Insertion of the C. porcellus CHK2 sequence into our alignment and comparison with the other placental mammalian CHK2 sequences revealed 34 C. porcellus-specific amino acid substitutions . We then scored these residues with Align-GVGD as if they were amino acid substitutions in the human CHEK2 sequence. All 34 scored C0, the most evolutionarily likely grade and the grade that contributes least to the difference that we observe between breast cancer patients and controls. Simulating and scoring all possible single-nucleotide substitutions to the canonical human CHEK2 cDNA sequence, we found that 57.2% of possible missense substitutions are C0. Taking into account differing probabilities of these substitutions due to their underlying sequence contexts as estimated by dinucleotide substitution rate constants [C. porcellus-specific substitutions would be C0 is ~0.58634 = 1.3 × 10-8. Thus selection acts against the rMSs of grade >C0. As these grades have sequentially increasing leverage (toward C65) on the test for trends, evolutionarily unlikely rMSs indeed drive the observed difference between patients and controls.One question that arises from this approach to missense substitution analysis is whether the rMSs that drive the difference between patients and controls are truly evolutionarily unlikely, which is shorthand for "subject to purifying selection such that they are disproportionately unlikely ever to become fixed as major alleles." To address this question, we waited until after our primary protein multiple sequence alignment had been created and the rare human missense substitutions had been scored, then we assembled an additional mammalian onstants , 58.6% oPtrend = 8.8 × 10-5) as opposed to 0.26/grade (Ptrend = 1.1 × 10-4) with the alternative approach. The important point is that the data were less compatible with chance when combined than when they were considered as either T+SJVs or rMSs alone.Looking forward to candidate gene studies, it could be useful to combine evidence from both T+SJVs and rMSs. The loglinear OR trend test provides a simple mechanism by which to achieve this end: observations of T+SJVs can either be combined with observations of the highest grade of missense substitutions (C65s) or we can add an eighth (even higher) carrier grade for the T+SJVs. For this data set, combining T+SJVs and C65 rMSs in grade 7 appeared to be slightly more effective: lognormal OR increased by 0.29/grade  or familial relative risks, the data do provide a basis on which to compare the relative contributions of these two classes of variants. Working from the control carrier frequencies and the OR point estimates  observed from the population-based Breast CFR sample series, we calculate attributable fractions of 0.014 for T+SJVs as compared with 0.015 for the sum of C15-C65 rMSs. In addition, we calculate a familial relative risk among first-degree relatives of 1.036 for T+SJVs as compared with 1.033 for a product across the C15-C65 rMSs. Thus, as a first approximation, the attributable fractions and familial relative risks of truncating variants and rare missense substitutions are virtually identical. It is important to remember that these attributable fraction and familial relative risk point estimates are inflated compared with those that would be obtained from a population-based study that included patients diagnosed in their 70s or older. In addition, as more than 25% of the T+SJVs observed in this study were nonsense and frame shift mutations other than c.1100delC, these data also speak to the importance of full open reading frame mutation screening to observe the majority of genetically relevant sequence variants in this cancer susceptibility gene.Previous studies have shown that some Saccharomyces cerevisiae Rad53 complementation assay, Shaag et al. [p.P85L is equivalent to wild-type CHEK2. While Bell et al. [in vitro kinase function assay, both Bell et al. and Shaag et al. concluded that the allele is effectively neutral. Sodha et al. [p.R137Q allele and found that it encodes a protein with normal stability and normal response to DNA damage. Bell et al. [p.R137Q allele and found that it has normal kinase activity. In addition, Sodha et al. [p.R180H allele and found that it encodes a protein with slightly reduced stability but normal response to DNA damage. Thus existing functional assay results for these three variants are consistent with their being either neutral or at most weakly pathogenic. Wu et al. [et al. [p.I157T allele was comparable to the wild type. More recent studies have reported that the allele is at least partially defective in dimerization and autophosphorylation, binding and phosphorylating Cdc25, and binding BRCA1 [p.I157T and c.1100delC are both present at appreciable frequencies and have been subject to independent risk estimates, p.I157T does appear to confer increased risk of breast cancer, but the OR or penetrance associated with the missense substitution appears to be more modest than that associated with the frame shift c.1100delC [p.R117G allele encodes a functionally defective protein and is in all likelihood pathogenic. Thus, for the missense substitutions that were observed in our mutation-screening study and subjected to functional assays, there is a qualitative trend toward agreement between the Align-GVGD classification and the functional assay result, consistent with the trend in ORs that we observed across the Align-GVGD-defined ordered series of missense substitution grades. However, since concordant results between in silico assessments and functional assays are not yet considered sufficient for formal clinical classification of missense substitutions observed in BRCA1 and BRCA2 [CHK2 functional assays has reached the point at which concordant results from an in silico assessment and a functional assay would be sufficient for clinically relevant classification of a CHEK2 missense substitution.Several of the missense substitutions observed in this study have been subjected to functional assays in one or more published works. For the 14 missense substitutions that Align-GVGD scored C0 and which we would consequently predict to be neutral or nearly so, assay results have been reported for 4 . Using a g et al.  found thl et al.  found tha et al.  assayed l et al.  also assa et al.  assayed u et al.  found thu et al.  and Alig [et al.  found thng BRCA1 -62. In p1100delC . At the 1100delC ,56,62. Tnd BRCA2 -66, it dCHEK2 and other genes with similar mutation profiles. In the 2003 American Society of Clinical Oncology Policy Statement Update on Genetic Testing for Cancer Susceptibility, the second and third "indications for genetic testing for cancer susceptibility" were that "2) the genetic test can be adequately interpreted, and 3) the test results will aid in diagnosis or influence the medical or surgical management of the patient or family members at hereditary risk of cancer" (pp. 2398) [CHEK2 sequence variants is sufficient to justify a difference in cancer surveillance strategies [ATM [ATM and CHEK2 [The genetic results described in this work, combined with the above functional assay summary, have implications for potential clinical genetic susceptibility tests that might include p. 2398) . With rerategies -70. Howeies [ATM  point tond CHEK2 , it may in silico analysis of missense substitutions that has now become a standard component in the integrated evaluation of unclassified variants in BRCA1 and BRCA2 [BRCA1 and BRCA2 mutation-screening data and then demonstrated its effectiveness by an analysis of ATM case-control mutation-screening data [CHEK2 analysis presented here stands as a methodological confirmation of our approach to the inclusion of rare missense substitution data in case-control mutation-screening studies. The logistic regression test for trends that we used also provides a simple approach to combining evidence from rare missense substitutions with evidence from protein-truncating sequence variants to build a more complete and statistically powerful approach to assessing case-control mutation-screening data than would be afforded by either method alone. From a technological perspective, we can envision combining exon capture and massively parallel sequencing to extend case-control mutation screening to entire biochemical pathways and beyond. On the basis of our post hoc power calculations, at least 2,000 patients and 2,000 controls would be required for a whole pathway  study, and 3,300 patients and 3,300 controls would be required to undertake a whole-exome study. On the one hand, these numbers could be an underestimate because CHEK2 might be among the most important  of the intermediate-risk class of breast cancer susceptibility genes. On the other hand, it could turn out that a test based on observations of evolutionarily unlikely sequence variants has an intrinsically lower false-positive rate than anonymous marker GWASs and consequently would not require a full Bonferroni multiple testing correction to reasonably constrain the rate of false-positive results.The rare missense substitution analysis model combining Align-GVGD with the logistic regression test for trends grew out of the nd BRCA2 ,73. We ping data . Thus thCHEK2 shows that the gene harbors many different rare pathogenic sequence variants, a substantial proportion of which are missense substitutions. From a clinical perspective, the risk of breast cancer conferred by some pathogenic sequence variants in CHEK2 may be great enough to be of use in a clinical cancer genetics setting, and we note that the technical capability of offering a multigene breast cancer susceptibility testing panel at relatively low per gene laboratory cost is in place. Yet, our results with both CHEK2 and ATM suggest that such a test would create a severe burden of unclassified missense substitutions and that a large fraction of the genetically relevant risk would reside in those unclassified missense substitutions. Paradoxically, on the basis of the research perspective of susceptibility gene identification and characterization, this study validates our approach to the analysis of rare missense substitutions observed during case-control mutation screening and provides a method to combine data from protein-truncating variants and rare missense substitutions into a one degree of freedom per gene test.This case-control mutation-screening study of Breast CFR: Breast Cancer Family Registry; GWAS: genome-wide association study; IRB: institutional review board; OR: odds ratio; PCR: polymerase chain reaction; rMS: rare missense substitution; SNP: single-nucleotide polymorphism; T+SJV: truncating and splice junction variant; WGA: whole genome amplification.The authors declare that they have no competing interests.FLCK contributed to study design, led the laboratory team and helped to draft the manuscript. FL contributed to study design, led the data analysis and helped to draft the manuscript. FD contributed to the mutation screening and data analysis and helped to refine the laboratory platform. MV contributed to the sequence alignment and data analysis. CV was responsible for data management throughput for the project and helped to refine the laboratory platform. DB contributed to the sequence alignment and method for analysis of rare missense substitutions. GD contributed to the mutation screening and data analysis and helped to refine the laboratory platform. NF contributed to the mutation screening and data analysis and helped to refine the laboratory platform. SMC contributed to the mutation screening and data analysis and helped to refine the laboratory platform. NR contributed to the mutation screening and data analysis and helped to refine the laboratory platform. TND contributed to the sequence alignment and data analysis. AT contributed to statistical analyses and helped to draft the manuscript. GBB contributed to statistical analyses and helped to draft the manuscript. JLH was responsible for patients gathered through the University of Melbourne and helped to draft the manuscript. MCS contributed to study design and contributed to the management of samples obtained through the University of Melbourne. ILA was responsible for patients gathered through Cancer Care Ontario. EMJ was responsible for patients gathered through the Northern California Cancer Center . SVT was responsible for overall study design, contributed to data analysis and helped to draft the manuscript. All authors read and approved the final manuscript.Supplementary Tables S1 and S2. Supplementary Table S1: Missense, nonsense, frame shift, and splice junction variants. Supplementary Table S2: CHEK2 protein multiple sequence alignment characterization.Click here for file


Single nucleotide polymorphisms (SNPs) in microRNA-coding genes may participate in the process of carcinogenesis by altering the expression of tumor-related microRNAs. It has been suggested that two common SNPs rs2910164 in miR-146a and rs11614913 in miR-196a2 are associated with susceptibility to hepatocellular carcinoma (HCC). However, published results are inconsistent and inconclusive. In the present study, we performed a meta-analysis to systematically summarize the possible association between the two SNPs and the risk for HCC.We conducted a search of case-control studies on the associations of SNPs rs2910164 and/or rs11614913 with susceptibility to HCC in PubMed, EMBASE, ISI Web of Science, Cochrane Central Register of Controlled Trials, ScienceDirect, Wiley Online Library and Chinese National Knowledge Infrastructure databases. Data from eligible studies were extracted for meta-analysis. HCC risk associated with the two polymorphisms was estimated by pooled odds ratios (ORs) and 95% confidence intervals (95% CIs). 5 studies on rs2910164 and 4 studies on rs11614913 were included in our meta-analysis. Our results showed that neither allele frequency nor genotype distribution of the two polymorphisms was associated with risk for HCC in all genetic models. Similarly, subgroup analysis in Chinese population showed no association between the two SNPs and the susceptibility to HCC.This meta-analysis suggests that two common SNPs rs2910164 and rs11614913 are not associated with the risk of HCC. Well-designed studies with larger sample size and more ethnic groups are required to further validate the results. Primary liver cancer is the sixth most common cancer worldwide . Due to its poor prognosis and high fatality rates, the incidence and mortality rates are almost equal. Primary liver cancer causes 695,900 deaths every year, making it the third most common cause of cancer-related deaths MicroRNAs (miRNAs) are small non-coding, single-stranded RNA molecules with typical length of ∼22 nucleotides. Functioning as post-transcriptional regulators, miRNAs complementarily bind to target mRNAs and negatively regulate their stability and transcriptional efficiency Single nucleotide polymorphisms (SNPs) in miRNA-coding genes may have effects on either the expression or the function of miRNAs by altering the secondary structure of miRNA precursors, consequently leading to the aberrant expression of a series of target genes and contributing to cancer susceptibility We carried out a publication search in PubMed, EMBASE, ISI Web of Science, Cochrane Central Register of Controlled Trials, ScienceDirect, Wiley Online Library and Chinese National Knowledge Infrastructure (CNKI) databases with the following search terms: (miR-146a OR miR-196a2 OR rs2910164 OR rs11614913) AND (hepatocellular carcinoma OR liver cancer OR hepatoma), by two independent investigators . Publication date and publication language were not restricted in our search. Reference lists were examined manually to further identify potentially relevant studies. We contacted the corresponding authors of conference abstracts without sufficient data for additional information by e-mail. If no reply or the author refused to provide the data required in this meta-analysis, the study was excluded. All studies matching the inclusion criteria were retrieved for further examination and data extraction. Investigators include experts in hepatobiliary surgery, biologists and qualified graduate researchers. All of the investigators have received training in literature search, statistics and evidence-based medicine.Studies included in our meta-analysis had to meet all the following criteria: (1)evaluated the associations between the SNPs miR-146a rs2910164 and/or miR-196a2 rs11614913 and susceptibility to HCC, (2) studied on human beings, (3)in a case-control design, (4)detailed genotype data were provided for the calculation of odds ratio (OR) and 95% confidence interval (95% CI), (5)if serial studies of the same population from the same group were reported, the latest study was included. We assessed the methodological qualities of included studies by the description of study population, detailed genotyping methods, the set of controls and cases and related statistical methods. Included studies had to meet the following criteria regarding methodology: (1) participants recruited from comparable populations with similar demographic background in control and case groups, (2) proper diagnostic criteria to determine the disease status, (3) Hardy-Weinberg equilibrium (HWE) reached in control population, (4) proper method to determine the genotypes with detailed description of method used, (5) appropriate statistical analysis performed, (6) detailed allele and genotype frequencies reported.Two investigators (Zhongxia Wang and Yin Cao) independently extracted data from the included studies. Data extracted from eligible studies included the first author’s name, publication date, country origin, ethnicity, genotyping method, total numbers of cases and controls and genotype frequencies of cases and controls. The two investigators checked the data extraction results and reached consensus on all of the data extracted. If different results were generated, they would check the data and have a discussion to come to an agreement. Three senior investigators  were invited to the discussion if disagreement still existed.P<0.05 was considered representative of a departure from HWE. ORs and 95% CIs were calculated to assess the strength of the association between miR-146a rs2910164 or miR-196a2 rs11614913 polymorphism and susceptibility to HCC. Pooled ORs were calculated for allele frequency comparison , additive model , dominant model , and recessive model , respectively. The significance of pooled ORs was determined by Z-test and P<0.05 was considered as statistically significant. Statistical heterogeneity among the studies was checked by chi-square-based Q-test. A P-value greater than 0.10 for Q-test indicates no significant heterogeneity existed among studies P value of Egger’s test less than 0.05 was considered representative of statistically significant publication bias For each study, HWE was evaluated using the goodness-of-fit chi-square test. et al. A total of 367 articles were retrieved after first search in PubMed, EMBASE, ISI Web of Science, Cochrane Central Register of Controlled Trials, ScienceDirect, Wiley Online Library and CNKI databases. As shown in Q-test in all of the genetic models showed no significant heterogeneity. Therefore, the pooled ORs were calculated using fixed-effects model. No significant association between miR-146a rs2910164 polymorphism and susceptibility to HCC was identified in any of the genetic models .The association between miR-146a rs2910164 polymorphism and susceptibility to HCC was analyzed in five independent studies with 1,912 cases and 2,149 controls. Results of the meta-analysis are shown in P = 0.088; GC versus GG: OR = 0.911, 95% CI 0.754–1.101, P = 0.336; CC versus GG: OR = 0.845, 95% CI 0.693–1.029, P = 0.095; GC/CC versus GG: OR = 0.883, 95% CI 0.739–1.056, P = 0.173; CC versus GC/GG: OR = 0.904, 95% CI 0.786–1.039, P = 0.155)  .P = 0.182; TC versus TT: OR = 1.121, 95% CI 0.855–1.470, P = 0.408; CC versus TT: OR = 1.318, 95% CI 0.881–1.974, P = 0.180; TC/CC versus TT: OR = 1.200, 95% CI 0.874–1.647, P = 0.260; CC versus TC/TT: OR = 1.132, 95% CI 0.965–1.326, P = 0.127). The results are summarized in 4 studies involving 1,790 cases and 1,635 controls were assessed for the association between miR-196a2 rs11614913 polymorphism and HCC risk. Significant statistical heterogeneity was identified in the comparison of allele frequency, TC versus TT, CC versus TT and in the dominant model so that random-effects model was used in these models. Fixed-effects model was used in the recessive model. However, for rs11614913, none of the genetic models produced significant association between rs11614913 polymorphism and HCC risk .P>0.05).Funnel plot and Egger’s test were performed to assess the publication bias of the literature. Symmetrical funnel plots were obtained in both of the SNPs tested in all of the models. Egger’s test further confirmed the absence of publication bias in this meta-analysis  to 0.709 , indicating no significant difference of susceptibility to HCC between CC and GG genotypes. Similarly, synthesis of the data with HBV positive control population obtained a pooled OR without statistical significance . The consistency of the stratified analysis suggests that HBV infection status in control populations may not be the factor that affected the stability of the comparison between CC and GG genotypes. Caution should be made when interpreting the result of the comparison between CC and GG genotype.Every single study involved in this meta-analysis was deleted each time to examine the influence of the individual data set to the pooled ORs. For rs2910164, the study from Zhang P = 0.01), indicatThe research on miRNAs has led to novel insights into the molecular mechanism of HCC miR-146a rs2910164 is located in the stem region opposite to the mature miR-146a sequence. This G>C polymorphism results in a change from G:U pair to C:U mismatch in the stem structure of miR-146a precursor. Several case-control studies have investigated the association between miR-146a rs2910164 polymorphism and risks of various cancers.et al.P = 0.10) to 0.71 . Zhang et al’s study with a relatively large sample size may be the source of potential heterogeneity or bias in the comparison between genotype CC and GG and could cause a major influence on the estimation of the association between rs2910164 polymorphism and HCC risk. Since HBV infection is a well-established risk factor for the development of HCC In this meta-analysis, a total of 5 case-control studies were analyzed to provide a comprehensive assessment of the association between miR-146a rs2910164 polymorphism and HCC. Our results did not support a genetic association between rs2910164 and susceptibility to HCC. Neither allele frequency nor genotype distribution was significantly associated with susceptibility to HCC. Since the incidence of gene polymorphisms may vary between different ethnic groups and this variation may interfere with the detection of minor effect of SNPs on HCC risk, subgroup analysis in Chinese population was performed to further explore the potential association between rs2910164 and the risk of HCC. However, even within the same ethnic group, no association of statistical significance was observed. Sensitivity analysis showed that the study from Zhang Another SNP shown to have potential relationship to the risk for HCC is rs11614913 in miR-196a2. It is reported that C allele of rs11614913 increased the expression of mature miR-196a2 in HCC tissues 4 studies on the relationship between rs11614913 and susceptibility to HCC were included in our meta-analysis. To our surprise, we failed to find any association between rs11614913 polymorphism and the risk for HCC in any of the examined genetic models. Similarly, subgroup analysis in Chinese population also showed no association of miR-196a2 rs11614913 polymorphism with susceptibility to HCC. Sensitivity analysis found no significant influence of any single study on pooled ORs, indicating the stability of this meta-analysis was acceptable.Several previous meta-analyses systematically reviewed the potential association of the two common polymorphisms rs2910164 and rs11614913 with susceptibility to cancer et al.To the best of our knowledge, this is the first meta-analysis evaluating the potential association between two common polymorphisms rs2910164 in miR-146a and rs11614913 in miR-196a2 and susceptibility to hepatocellular carcinoma. However, caution should be made when interpreting the results due to some limitations of this meta-analysis. Firstly, heterogeneity was detected in some comparisons of rs11614913 and sensitivity analysis showed that the study from Zhang In summary, despite the limitation, this meta-analysis suggests that miR-146a rs2910164 and miR-196a2 rs11614913 polymorphisms may not be associated with the risk of HCC. Well-designed studies with larger sample size and more ethnic groups should be considered to further clarify the association. Moreover, other factors such as gender, age, HBV/HCV infection status and alcohol consumption should also be considered in future studies.Table S1MOOSE checklist.(DOC)Click here for additional data file.


S-transferase Mu-1 (GSTM1) and Glutathione S-transferase theta-1 (GSTT1) in the detoxification of environmental carcinogens, environmental toxins, and oxidative stress products, genetic polymorphisms of these two genes may play important roles in the susceptibility of human being to hepatocellular carcinoma. However, the existing research results are not conclusive.Due to the possible involvement of Glutathione A systematic literature search using databases  for the eligible studies meeting the inclusion criteria including case-control studies or cohort studies is evaluated using an updated systematic meta-analysis.GSTM1 null genotype  and GSTT1 null genotype . Analysis using the random-effects model found an increased risk of HCC in GSTM1-GSTT1 dual null population . In addition, subgroup analyses showed a significant increase in the association of GST genetic polymorphisms  with HCC in southeast and central China mainland. However, available data collected by this study fail to show an association between GST genetic polymorphisms and HCC in people from the Taiwan region . Sensitivity analysis and publication bias diagnostics confirmed the reliability and stability of this meta-analysis.Significant increase in the risk of HCC in the Chinese population is found in GSTM1 and GSTT1 null genotypes are associated with an increased HCC risk in Chinese population. Peoples with dual null genotypes of GSTM1-GSTT1 are more susceptible to developing HCC. In conclusion, GST genetic polymorphisms play vital roles in the development of HCC in the Chinese population.Our results indicate that both  Due to a high mortality, hepatocellular carcinoma (HCC) is one of the most serious health problems worldwide S-transferases (GSTs) family is an important phase II isoenzyme which can detoxify environmental carcinogens and toxins, oxidative stress products, and modulate the induction of other enzymes and proteins in the cell at the same time GSTs family are composed of many cytosolic, mitochondrial, and MAPEG proteins. Human GSTs can be divided into eight main classes including alpha, mu, pi, theta, sigma, kappa, omega and zeta GSTM1 and GSTT1  both play important roles in human carcinogenesis. Epidemiologic investigations related to genetic association including case-control and cohort studies suggested the association between GST genetic polymorphisms and HCC risk. However, some of these studies with sparse data, unreasonable and highly underpowered designs, and differential in research methodology could all inevitably influence the robustness of their results. Meta-analysis can avoid these weaknesses by selecting all eligible studies and reducing random error. To identify the association of GST genetic polymorphisms with the susceptibility to hepatocellular carcinoma in the Chinese population, an updated systematic meta-analysis was performed in this study by using a full reference search (from January 1996 to October 2012) and a careful reinvestigation strategy.The Glutathione GSTM1 and/or GSTT1 polymorphisms with the susceptibility of HCC in China. Meanwhile, reference lists of the relevant articles were also collected. Search was performed through websites of http://www.baidu.com and http://scholar.google.cn to identify additional eligible studies. MeSH terms (“glutathione S-transferase” or “GST” or “GSTM1” or “GSTT1”) and (“hepatocellular carcinoma” or “liver cancer” or “HCC”) and (“China” or “Chinese” or “Taiwan”) were used in PubMed. These keyword retrieval strategies were also used in other databases. When there was more than one article published in a same case series, the latest and/or the comprehensive one would be adopted only. Eligible research articles not captured by above research strategies would be further searched by bibliographies.A computerized literature search was carried out in Embase, PubMed, Scopus, Chinese Biomedical Database (CBM), CochraneLibrary, Chinese National Knowledge Infrastructure (CNKI), and Wanfang Data (the latest research was retrospected to October 2012) to collect articles with case-control or cohort studies related to the association of GST genetic variances and HCC risk included these owning either with a balance match or not; (2) in the Chinese population; (3) the articles provided raw data including odds ratio (OR) with 95% confidence interval (CI) and respective variance, or the relevant information could be calculated.Inclusion criteria are: (1) case-control and cohort studies, in which individuals or samples used for evaluation of the association between Exclusion criteria are: (1) raw data not available for retrieval; (2) multiple articles based on a same population and published by a same research team, only the latest and/or the largest population study was adopted, others would be excluded; (3) meeting abstract, case reports, editorials, review articles and other meta-analysis were exclusive.GSTM1, GSTT1 and null of both genotypes in cases and controls.To decide inclusively or exclusively, articles were identified by two independent reviewers using a standardized data extraction form designed by our group. Data with discrepancies in identification were discussed. If consensus was not achieved, the decision was made by a third reviewer. Both title and abstract from all potential included articles were screened to identify their relevance. Additionally, if title and abstract were ambiguous, full articles were also investigated. The following information was collected from each study: first author, year of publication, geographical location, study time, pathologic diagnosis, source of control, characteristic of cases and controls, and genotype frequency of null P<0.05 considered statistically significant; (2) Statistical heterogeneity among studies was assessed with the Q and I2 statistics 2 were claimed to test the variation which was due to heterogeneity or by random error P value of heterogeneity tests was no more than 0.1 (P≤0.1), we used random effects model. When P value of heterogeneity test was more than 0.1 (P>0.1), we used fixed effects model vs. ≥100), source of control ; (7) All analyses were performed using the software State version12.0 , Review Manager 5.0 . All the P values were two sided.(1) The pooled OR and 95% CI were determined by Z test with GST genetic polymorphisms and HCC risk by both Chinese and English database GSTM1 (3712 cases and 6024 controls), 21 articles of GSTT1 (3378 cases and 5400 controls) and 12 articles of both GSTM1 and GSTT1 (1562 cases and 2537 controls) are shown in We ultimately identified a total of 27 articles reporting the relationship between GSTM1 null genotype and HCC susceptibility. 12 articles were published in Chinese and 14 articles in English. Results obtained from a random-effects model showed a significant association between the GSTM1 null genotype and HCC risk in the Chinese population . The forest plot was showed in 26 articles  including 3712 cases and 6024 controls were investigated in this study to evaluate the association between GSTT1 null genotype and HCC susceptibility. 9 articles were published in Chinese and 12 articles were published in English. Results showed that the GSTM1 null genotype was significantly associated with HCC risk demonstrated by random-effects model in the Chinese population . The forest plot was shown in 21 articles including 3378 cases and 5400 controls were used for the investigation of the association between GSTM1-GSTT1 null genotype and HCC susceptibility. Results indicated that dual-null genotype of GSTM1-GSTT1 also had a significant association with HCC risk in the Chinese population . The forest plot was shown in 12 articles (6 articles in Chinese and 6 articles in English) including 1763 cases and 2537 controls were used to evaluate the relationship between P values for GSTM1, GSTT1, and the interaction of GSTM1-GSTT1 were all less than 0.001, I2 values were 77.4%, 71.1%, and 77.7%, respectively). In this meta-analysis, subgroup analyses contained geographical location , case number (<100 vs. ≥100), source of control . The between-study heterogeneity showed that the major source of heterogeneity came from China mainland population. Association between GST genetic polymorphisms and HCC risk increase was significant in subgroup analyses of both southeast and central regions in China mainland population, but no significant in Taiwan population. Other subgroup analyses results were shown in The substantial between-study heterogeneity of the three above analyses were observed . Galbraith plots  still existed. These results were shown in Sensitivity analysis was performed by sequential excluding one article each time. The significance of all ORs was not changed. We used Galbraith plot to omit some possible major sources of heterogeneous articles. The results were showed in th plots  spotted GSTM1, GSTT1, and dual-null genotype of GSTM1-GSTT1 .Beggar’s funnel plots and Egger’s publication bias plots were used to assess the potential publication bias for M1-GSTT1 . No publGST genetic polymorphisms and HCC risk are inconsistent according to the present research results. This may be caused by several reasons. Improper matching or insufficient case and control numbers used in the studies are all possible reasons. One meta-analysis GST genetic polymorphisms and HCC risk didn’t cover all conclusive articles published in Chinese and English databases. In this meta-analysis paper, overlapped data was found in two adopted studies GST genetic polymorphisms and HCC risk have emerged since these two meta-analysis papers were published.The association between GST genetic polymorphisms and susceptibility to HCC in the Chinese population, we performed an updated systematic meta-analysis. In this study, 27 articles (3781 patients and 6104 controls) were selected from Chinese and English databases. 26 studies (3712 cases and 6024 controls) out of the 27 articles were used for investigation of the relationship between GSTM1 null genotype and HCC susceptibility. 21 studies (3378 cases and 5400 controls) out of the 27 articles were used to evaluate the relationship between GSTT1 null genotype and HCC susceptibility. 12 studies (1763 cases and 2537 controls) were applied for evaluation for the GSTM1-GSTT1 gene. Random-effects model of meta-analysis shows significant associations of polymorphisms of GSTM1 null gene , GSTT1 null gene , and GSTM1-GSTT1 dual null gene , respectively, with HCC risk in the Chinese population. Subgroup analyses on GSTM1 null gene indicate that geographical location , case numbers and source of controls are significantly associated with HCC risk. Results of subgroup analyses on GSTT1 null gene and GSTM1-GSTT1 dual null gene indicate that geographical location , case numbers  and source of controls are also significantly associated with HCC risk. Reasons for inconsistent in conclusions between China mainland and Taiwan region may be caused by environmental factors. Moreover, limited investigative numbers of the case-control/followed up studies from Taiwan region may result in difficulty for getting stable risk estimation, though these investigations own low between-study heterogeneity. In addition, studies with case number less than 100 may have effects on drawing a proper estimation for the association between GST genetic polymorphisms and HCC risk. Therefore, further well design case-control/followed-up studies, especially with a larger case number, are necessary to provide better evidences for the evaluation. Heterogeneity analysis is a key part of meta-analysis. Q statistic test (Cochran’s Q statistic) and I2 statistic test are commonly used to test and quantify the between-study heterogeneity. The major source of heterogeneity in the China mainland population detected in the subgroup analysis might come from the environmental difference which could affect their sensitivity to particular genomic variants. In this meta-analysis, Galbraith plot was performed for identifying the articles with possible heterogeneity. However, in the analyses on the GSTM1 polymorphism and HCC risk, we kept several articles with obvious between-study heterogeneity because too many articles omitting could cause some biases. For the association of GSTT1 null gene and HCC risk, we deleted 3 articles GSTM1-GSTT1 gene . Therefore, we applied the random-effects model to evaluate the pooled OR for GSTT1 and GSTM1-GSTT1 genes, respectively. After the above adjustments, the associations were increased between GSTT1 and GSTM1-GSTT1 polymorphisms and HCC risk . In this study, Beggar’s funnel plots and Egger’s linear regression test were applied to assess the potential publication bias. No publication bias was detected .To evaluate the association of GST genetic polymorphisms are associated with the susceptibility to several carcinomas. Takahiko Katoh et al.GSTM1 null genotype might be associated with susceptibility to gastric adenocarcinoma and distal colorectal adenocarcinoma in Japanese population. Wang J et al. GSTM1 null and GSTP1 Val was significantly associated with an elevated lung adenocarcinoma risk . Helzlsouer K J et al. GST gene family might be associated with an increased susceptibility to breast cancer . Compared to the control group value of 41.8%, Zhong S et al. GSTM1 gene null individuals in colorectal cancer group. Our meta-analysis results demonstrate that there is an association between GST genetic polymorphisms and susceptibility to HCC in the Chinese population. Thus, further epidemiological and molecular biological studies are necessary to clarify the role of GST genetic polymorphisms in HCC and other carcinomas.Research evidences suggest that Nevertheless, there were several limitations to this meta-analysis. (1) Observational studies were susceptible to various biases such as selection bias. Due to some studies without clear explanation for the pathologic diagnostic results of all/part subjects , therefoGST genetic polymorphisms are associated with the increased risk of HCC in the Chinese population. To further evaluate gene-to-gene and gene-to-environment combined effects on GST genetic polymorphisms and HCC,both large scale multicenter epidemiological studies in total population and/or selected population with different environmental background are urgently needed.In summary, our results suggest


MDM2 polymorphism (rs2279744) in PVR. Distribution of T309G MDM2 genotypes among European subjects undergoing RD surgery was evaluated. Proportions of genotypes between subsamples from different countries were analyzed. Also, a genetic interaction between rs2279744 in MDM2 and rs1042522 in p53 gene was analyzed. Significant differences were observed comparing MDM2 genotype frequencies at position 309 of intron 1 between cases  and controls . The proportions of genotypes between sub-samples from different countries showed a significant difference. Distribution of GG genotype revealed differences in Spain (35.1–53.0)/(22.6–32.9), Portugal (39.0–74.4)/(21.4–38.9), Netherlands (40.6–66.3)/(25.3–38.8) and UK (37.5–62.4)/(23.3–34.2). The OR of G carriers in the global sample was 5.9 (95% CI: 3.2 to 11.2). The OR of G carriers from Spain and Portugal was 5.4 (95% CI: 2.2–12.7), whereas in the UK and the Netherlands was 7.3 (95% CI: 2.8–19.1). Results indicate that the G allele of rs2279744 is associated with a higher risk of developing PVR in patients undergoing a RD surgery. Further studies are necessary to understand the role of this SNP in the development of PVR.Proliferative vitreoretinopathy (PVR) is still the major cause of failure in retinal detachment (RD) surgery. It is believed that down-regulation in the p53 pathway could be an important key in PVR pathogenesis. The purpose was to evaluate the impact of T309G  Proliferative vitreoretinopathy (PVR) is still the major cause of failure in retinal detachment (RD) surgery Although PVR was identified in 1983 as an independent entity Most research has to date attempted to identify the clinical risk factors related to the development of PVR after RD; however, these clinical factors do not completely explain the probability of its onset Besides the role of many inflammatory mediators in the development of PVR and RD In normal unstressed cells, p53 is a very unstable protein with a half-life ranging from 5 to 30 min, which is present at very low levels owing to continuous degradation largely mediated by murine double min 2 protein (MDM2) Recently, it has been reported that the levels of p53 expression could be a checkpoint in the development of RD and PVR, and how its local increase in the vitreous by using inhibitors of MDM2, seem to be a promising approach as a prophylaxis in experimental RD and also in experimental PVR MDM2 gene is a key negative regulator of p53 and in humans seems to have two promoter-enhancer regions that regulate the levels of MDM2 mRNA. The first promoter is 5′ to the first exon and likely regulates the basal level of MDM2 in a nonstressed cell. The second promoter region is in the first intron and this region increases the expression of MDM2 after a p53 response The MDM2 T309G polymorphism in the first intron of MDM2 gene, in a consecutive sample of patients undergoing primary rhegmatogenous RD surgery with and without PVR, recruited from several European clinical centres through the project named Retina 4.Thus, the purpose of this study was to analyze the distribution of the The study was approved by the local Ethics Committees of Instituto de Oftalmobiología (IOBA-Retina Group) , Hospital San João , Moorfields Eye Hospital  and Rotterdam Eye Hospital  and followed the tenets of the Declaration of Helsinki. All patients received written informed consent before entering in the study.MDM2 gene was carried out. Second, the interaction between this polymorphism and the p53 codon 72 polymorphism (rs10425229) was investigated.DNA samples from the Retina 4 project were analyzed. This work is made up in two steps: first, a candidate gene association study in the T309G polymorphism (rs2279744) located into the The association study was carried out among 555 patients from 7 centers: 3 in Spain, 2 in Portugal, 1 in the United Kingdom (UK) and 1 in Netherlands. The global sample was divided in sub-samples according to the country for the analysis. This study was carried out in two phases. In the first one, sub-samples from Spain and Portugal were analyzed. After significant results were found in this first cohort, subsequent samples from the UK and the Netherlands were analyzed (second phase). To compare if there were differences regarding geographical localization in the odds ratio analysis, Spain and Portugal were considered as Southern countries and the UK and the Netherlands as Northern countries. Genotypic and allelic frequencies were also compared between cases and controls in the global series., according to Machemer classification, were included as cases.Detailed explanation of the exclusion and inclusion criteria for classification of patients has been provided in a previous publication MDM2 polymorphism (rs2279744) and one SNP (rs1042522) located in the p53 gene previously identified by our group as significantly associated to PVR p53 polymorphism were same samples than for the MDM2 SNP. Carriers of Pro variant of rs1042522 and G variant of rs2279744, were analyzed in the global sample and in the sub-samples from different countries.Interaction between the MDM2 T309G polymorphism was performed at the Molecular Medicine Unit at the University of Salamanca,  blinded to the clinical status of patients, using the PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) technique Genotyping of the MDM2 T309G polymorphism was detected after amplification of genomic DNA with the forward primer 5'-GAGGTCTCCGCGGGAGTTC-3' and the reverse primer 5'- CTGCCCACTGAACCGGC-3'.MDM2 intron 1 was amplified within a 155 pb DNA fragment that was digested with the restriction endonuclease MspA1I .The 2, 50 µM of each dNTP and 1.25 U of HotMaster Taq DNA polymerase . DNA was amplified with the following steps: an initial 5-min denaturation at 94°C, followed by 35 cycles of 94°C for 30 s, 61°C for 30 s, 72°C for 30 s, and a final elongation at 72°C for 10 min.PCR reactions were performed in a 50-µl reaction mixture containing 100–200 ng of target DNA, 20 pmol of each primer, 2.5 mM MgClThe resulting fragments were separated on 3.5% agarose gel  and the The polymerase chain reaction fragments, containing T and G alleles, after digestion migrated as 2 fragments of 101 and 54 bp for G homozygotes (GG), 1 fragments of 155 for T homozygotes (TT), and 3 fragments of 157, 101 and 54 bp for heterozygotes (TG).The quality of data was evaluated in control sub-samples by Hardy-Weinberg equilibrium using the Chi-square test. Genotypic frequencies were estimated in each sub-sample. The proportions of genotypes and the G homozygote groups between sub-samples were analyzed. Also, the genotypic and allelic frequencies were compared between cases and control in the global sample and in the sub-samples from different countries. In the genetic interaction, patients carrying the Pro variant of rs1042522 and the G variant of rs2279744 were analyzed in the global sample and in the sub-samples from different countries.Association was investigated using the Chi-Square and the Fisher’s tests. The strength of association was measured using Odds Ratio (OR) and 95% confidence intervals (CI). Two inheritance models were considered: co-dominant model that allows every genotype to give a different and non-additive risk, and recessive model in which two copies of the G allele are necessary to change the risk. The Akaike Information Criterion (AIC) In order to adjust p-values for multiple comparisons a permutation test was performed. We used 1000 random shuffles of the case/control labels to get the correct distribution of test statistics under the no-association hypothesis. The ranking of the real test statistic among the shuffled test statistics gives the adjusted p-values.A total of 555 peripheral DNA blood samples including 134 cases and 421 controls were analyzed (203 from Spain (36.57%), 68 from Portugal (12.25%), 121 from Netherlands (21.80%) and 163 from the UK (29.36%). Regarding clinical information some significant associations were observed. The control group was significantly older than cases (p<0.0001) with the difference between median of 6 years (95% CI: 3.39-8.31). A significant association in patients with history of PVR in the fellow eye was found in the cases group. Also status of the lens was determined because aphakia has been related to a higher incidence of PVR There were no failures for the genotyping process, with a global call rate of 96.21%. Additionally, in order to ensure accuracy of allele-specific results, a randomized selection of samples PCRs were assessed by an independent researcher unaware of the patients’ status. All control sub-samples verified the Hardy-Weinberg equilibrium.Phase I: genotypic distribution of MDM2 T309G polymorphism in Spain and Portugal.The frequencies of the genotypes in each country are shown in The odds ratio of G carriers from Spain and Portugal together considering a co-dominant model  (AIC =  307.7 vs 311.4 of a recessive model) was 5.4 (95% CI: 2.3 to 12.7) .Phase II: genotypic distribution of MDM2 T309G polymorphism in UK and the Netherlands.The frequencies of the genotypes in patients from the UK and the Netherlands are shown in When all samples were grouped,  significSignificant differences in the analysis of the allelic frequencies were found between cases and controls in Spain and Portugal and between cases and controls in UK and Netherlands p53 and MDM2 genotypes (Pro variant plus G variant carriers)  were found. A significant difference in the distribution of genotypes between the controls and cases (p<0.0001) was found in the global sample. The odds ratio of Pro and G carriers in global sample was 10.19 (95% CI: 3.2 to 31.9)  who meet both to 31.9) .Significant differences in the distribution of genotypes between the controls and cases from Spain and Portugal together (p = 0.0006) and UK and Netherlands together (p<0.01) were found. The odds ratio of Pro and G carriers from the UK and the Netherlands together was 6.5 (95% CI: 1.5 to 28.2), whereas, the odds ratio of Pro and G carriers from Spain and Portugal together was 20.4 (95% CI: 2.4 to 169.5) .Our results show that Spanish and Portuguese carriers of the homozygous G SNP at position 309 have a 5.4 -fold increased risk of PVR after RD than those that carry the T allele. This observation was confirmed also in Dutch and British population (odds ratio 7.3 (95% CI: 2.8 to 19.1). Interestingly, when Pro and G restriction site carriers in the global sample were analyzed, an additional effect (odds ratio 10.19 (95% CI: 3.2 to 31.9) was found. But both polymorphisms were only present in 16 patients out of 555 patients. Although significant results were also found in the latter analysis, we consider that 16 may be too small a sample to draw absolute conclusions and further studies may be necessary in order to know the absolute potential risk of this restriction site association.PVR is considered a multifactorial disease Apoptosis is critically important during various developmental processes, it is necessary to rid the body of pathogen-invaded cells and also is involved in the removal of inflammatory cells and the evolution of granulation tissue into scar tissue As mentioned, PVR is considered an abnormal wound-healing process induced by the production of a retinal break and vitreal escape of RPE cells into an appropriate intraocular pro-inflammatory environment Current studies have highlighted the involvement of extrinsic and intrinsic pathways of apoptosis in retinal cells after RD, and also, the existence of other pathways, such as programmed necrosis, when apoptosis are inhibited p53 Arg72Pro polymorphism and PVR has been recently reported by our group p53 gene, which are associated with a decrease in apoptotic function of p53, have higher risk of PVR after RD. Furthermore it has been reported that suppression of p53 expression might be a necessary event in the development of RD and PVR, and maintaining levels of p53 with agents such as Nutlin-3, which prevents the interaction between p53 and MDM2, might be effective in the prophylaxis of RD and also PVR vitreous-induced contraction An association between MDM2 T309G polymorphism affects binding of transcription factor Sp1 and is associated with an increased expression of the MDM2 mRNA and attenuation of the p53 pathway The Also, it has been reported that in contrast to other retinal cell types, the RPE cells are resistant to apoptosis, to TNFA and oxidative stress, which trigger apoptosis in wide range of cells MDM2 T309G polymorphism has been associated to several cancers p53 (i.e. p53 codon 72 polymorphism) increases the risk of development of several tumors The MDM2 gene, associated with a decrease in apoptotic function of p53, have higher risk of PVR after RD. We can speculate that the reduction in the levels of apoptosis in retinal cells may activates other cell death pathways, like programmed necrosis, which would increase the intraocular inflammation after RD, thus generating a cascade of tissue responses that generate and amplify the hostile microenvironment in which activated RPE can trans-differentiate. It is also possible that the decline in the levels of p53 is a crucial checkpoint in RD and PVR, and the recently developed ocular formulation of Nutlin-3, which can be administered by sub-conjunctival injection Results of this work suggest that carriers of the G allele of the MDM2 gene that shows a strong association with PVR across several groups. Although we have carried out the study in two phases these findings must be interpreted with caution until these results are confirmed with further replication studies in order to confirm its association, because one of the major pitfalls of genetic association studies are the false positives This study had some limitations. One important issue in an association study is the sample size In summary, this study indicates that the p53 pathway could be implicated as a significant risk factor for PVR after RD and also it highlights the role of these SNPs (rs1042522 and rs2279744) as possible markers of PVR risk.


SLC19A1, commonly known as RFC-1) has been associated with maternal risk of the birth of a child with Down Syndrome (DS), but results are controversial. We searched major online databases to identify available case-control studies, and performed a meta-analysis to summarize the data concerning this association. Nine independent case-control studies were identified for a total of 930 DS mothers (MDS) and 1240 control mothers. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using both fixed and random effects models. An increase in the risk of having a birth with DS was observed for carriers of the homozygous GG genotype , even after removal from the meta-analysis of published data with deviations from Hardy-Weinberg equilibrium (HWE) in controls . Moreover, the pooled OR under the fixed effects model showed an increase in the maternal risk for the G allele . Present results suggest that the maternal RFC-1 80A>G polymorphism might be associated with an increased risk of having a birth with DS, particularly among carriers of the GG genotype.A common polymorphism (c.80A>G) in the gene coding for the reduced folate carrier ( S-adenosylmethionine (SAM), and for the synthesis of DNA and RNA precursors .A meta-analysis of the selected studies , Table 2ts model  and the ts model . The Chi studies ,38, and g method ,37. The Sensitivity analyses were performed excluding the one study  with HWEp-value, resulting from χ2 analysis, indicated departure from the equilibrium only in one study [RFC-1 80A>G polymorphism and maternal risk of birth of a child with DS, and results are shown in vs. AG or AA), with an OR = 1.27 (95% CI: 1.04–1.57) if all the studies were evaluated, and an OR = 1.26 (95% CI: 1.02–1.55) after removal of the study with HWE deviations in controls. Similar results were obtained when removing both the one study with HWE deviations and the one in which control mothers had previous experience of miscarriage/abortion: OR = 1.33 (95% CI: 1.06–1.66). Since heterogeneity among studies was not significant, we used the fixed effects model to estimate the association; however, similar results were obtained also with the random effects model  gave significant results in the whole set of studies, with an OR = 1.14 (95% CI: 1.01–1.30), but this association was not significant after removal of the study with HWE deviations in controls, and again significant when removing both this study and the one in which control mothers experienced previous miscarriages/abortions (vs. AA), using both fixed or random effects estimates  for bortions . No signstimates .RFC-1 80A>G polymorphism in modulating maternal risk for having a child with DS, and particularly an increased maternal risk for carriers of the homozygous RFC-1 80GG genotype. Those data are in agreement with several previous studies observing an increased maternal risk for trisomy 21 in individuals carrying the RFC-1 80GG genotype either alone [RFC-1 genetic polymorphisms known to be in linkage disequilibrium with the 80A>G one have been associated with an increased risk for congenital heart defects in the DS offspring [Results of the present meta-analysis suggest a possible role for the er alone ,21 or coer alone ,22,24. Mer alone ,27,28. Iffspring , overallffspring . RFC-1 iffspring .vs. AA or AG). By contrast, some differences were observed concerning the allele contrast (G vs. A) model. Those differences can be explained taking into account both Q and 2I values and considering that under the fixed effects model, by definition, the overall estimates is more influenced by larger studies and information coming from small studies has a small weight, while in the random effects model, since each study is assumed to provide information about a different effect size, all these effect sizes are represented in the summary estimate [I2 was equal to 0, indicating that 0% of variation across studies was due to heterogeneity rather than chance, and explaining why we obtained very similar results using either a fixed or a random effects model. Conversely, under the allele contrast model heterogeneity among studies was still low, but the I2 value revealed that 2% of the variation across studies was due to heterogeneity. In this case 95% confidence intervals were wider under the random effects model than under the fixed effects one, leading to different outcomes.The odds ratios and 95% confidence intervals obtained using both fixed and random effects models were very similar under the recessive genetic model  that caused chromosome 21 nondisjunction [RFC-1 80GG genotype with increasing maternal age [RFC-1 80GG genotype [Albeit largely promising, present results deserve additional discussion and confirmation in subsequent well-designed studies. Among factors that must be taken into account when assessing the contribution of folate gene polymorphisms to the maternal risk for chromosome 21 malsegregation are maternal age and the use of folate supplements at periconception, as well as the type of meiotic error (junction ,41. A brjunction ,21,26, ojunction ,25,27, ajunction ,29. Despjunction ,16,17, srnal age , and othgenotype .Another factor deserving further investigation is the contribution of ethnicity. Given the paucity of available comparable case-control studies for each ethnic group included in the present meta-analysis , we did RFC-1 80GG genotype, suggesting that the studied polymorphism deserves further consideration. Additional case-control studies are required to clarify the joint effect of dietary factors, maternal age at conception, and the type of meiotic error, as well as the contribution of ethnic and geographic factors.The present meta-analysis of the literature reveals a significant increased maternal risk for having a birth with DS in carriers of the


We hoped to identify miRNA polymorphism or a combination of several polymorphisms that could serve as biomarkers for predicting the risk of gastric cancer and its precancerous diseases.The aim of this study was to investigate the interaction effects of pri-let-7a-1 rs10739971 with PGC rs4711690 C→G, PGC rs6458238 G→A, PGC rs9471643 G→C, and ERCC6 rs1917799 in 471 gastric cancer patients, 645 atrophic gastritis patients and 717 controls.Sequenom MassARRAY platform method was used to detect polymorphisms of pri-let-7a-1 rs10739971 G→A, ERCC6 rs1917799 polymorphism was observed for the risk of gastric cancer (Pinteraction = 0.026); and interaction effects of pri-let-7a-1 rs10739971 polymorphism with PGC rs6458238 polymorphism (Pinteraction = 0.012) and PGC rs9471643 polymorphism (Pinteraction = 0.039) were observed for the risk of atrophic gastritis.An interaction effect of pri-let-7a-1 rs10739971 polymorphism with ERCC6 and PGC polymorphisms could provide a greater prediction potential than a single polymorphism on its own. Large-scale studies and molecular mechanism research are needed to confirm our findings.The combination of pri-let-7a-1 rs10739971 polymorphism and  Individuals with similar living habits and living in similar environments possess different risks of cancer. Identifying and predicting individuals at high risk of developing cancer can indicate the need for changing living habits. Single nucleotide polymorphisms (SNPs) play a pivotal role in predicting individuals with increased cancer risk. In recent years, polymorphisms of pri-miRNA were reported to be biomarkers for predicting cancer risk, such as pri-miR-34b/c rs4938723 polymorphism Currently, investigators mainly focus on gene SNP-SNP interactions, which can cause protein-protein interactions, and few studies have investigated interactions between miRNA polymorphisms and gene polymorphisms, which can cause protein-RNA interactions. miRNA has been reported to take part in the multi-gene network of gastric carcinogenesis pepsinogen C (PGC) and excision repair cross complementing group 6 (ERCC6) with the risk of gastric cancer, and found that PGC gene rs4711690 C→G, rs6458238 G→A, and rs9471643 G→C polymorphisms had protective effects against atrophic gastritis ERCC6 rs1917799 T>G polymorphism in the promoter region was associated with increased gastric cancer risk in a Chinese population Our previous candidate gene association study investigated the association of PGC and ERCC6 polymorphisms in samples of the same group, and discuss its application prospects in gastric cancer and its precancerous diseases. To our knowledge, this is the first study attempting to assess potential SNP-SNP interactions of miRNA SNPs and gene SNPs at two or more loci implicated in gastric cancer susceptibility. We hoped to find combinations of gene-gene polymorphisms that could predict the risk of gastric cancer and its precancerous diseases, and to provide experimental evidence for the early diagnosis of gastric cancer.In this case-control study using a Chinese population, we investigated the association of pri-let-7a-1 rs10739971 with gastric cancer risk, and the interaction effects of miRNA-let-7a-1 rs10739971 polymorphism and In this study, a total of 1834 individuals, which included 471 gastric cancer patients, 646 atrophic gastritis patients and 717 controls, were retrospectively recruited from patients undergoing gastroscopy examination screening in Zhuanghe region and patients who underwent gastroscopy examination or gastric surgery at the First Affiliated Hospital of China Medical University, Liaoning Province, China between 2002 and 2011. Fasting venous blood was collected and all enrolled participants were diagnosed based on their gastroscopic and histopathological examinations. Gastric cancer was diagnosed based on WHO criteria, and atrophic gastritis and superficial gastritis were classified by Sydney's classification. Eligible controls were those participants with a normal stomach or only gastritis according to gastroscopic and pathological examinations and had no other diseases.The design of the study was approved by the Human Ethics Committee of China Medical University . Written informed consent was obtained from all participants. Medical histories  were obtained by questionnaire and the records were computerized. Each individual involved in the study provided written informed consent for epidemiological investigation. Detailed participant characteristics are summarized in g for 5 min to disperse the clot. Following clot disruption, 400 µL of TE, 25 µL of 10% SDS and 5 µL of 20 mg/mL proteinase K were added and incubated at 37°C overnight. The supernatant was extracted and an equal volume of phenol was added. The tube was placed on a rotator for 15 min and then centrifuged at 10,000 × g for 15 min. The supernatant was removed and a second extraction was performed with the addition of an equal volume of a mixture of phenol and chloroform (1∶1). Following centrifugation, the supernatant was removed and a third extraction was performed with the addition of an equal volume of chloroform. Following centrifugation, the supernatant was absorbed and two volumes of protein precipitation solution (two volumes of absolute ethanol containing 10% 3 mol/L sodium acetate) were added and incubated for 2 h at −20°C. Each sample was centrifuged at 10,000 × g for 10 min. After centrifugation, the resulting DNA pellet was rinsed with 75% ethanol and centrifuged at 10,000 × g for 10 min. The 75% ethanol was decanted and the tube inverted on clean absorbent paper for 30 min. The resulting DNA was reconstituted in TE buffer and stored at −20°C until use.Genomic DNA was extracted using a previously described method with slight modification DNA samples were diluted to working concentrations of 50 ng/µL before genotyping. The assays, primer design, and genotyping of 29 polymorphism sites selected for a candidate gene associated study were all carried out by CapitalBio  using Sequenom MassARRAY platform  between May 2011 and December 2011 based on the manufacturer's directionsH. pylori were performed to check the status of H. pylori infection using ELISA , as described previously H. pylori-IgG higher than 34EIU . Briefly, serum samples were diluted 1∶200 (5 µL + 995 µL) with diluent buffer and mixed well. Then 100 µL of blank solution, calibrators, controls and diluted samples were added to wells. The plate was covered with the incubation cover and incubated for 30 min at 37°C. After incubation, the wells were washed five times with 350 µL of diluted (1∶100) washing buffer and the plate gently tapped several times on filter paper. Then 100 µL of mixed conjugate solution was added to the wells and incubated for 30 min at 37°C. After incubation, the wells were washed again and 100 µL of mixed substrate solution was added to the wells before incubating for 30 min at room temperature (20–25°C) in a dark environment. Lastly, 100 µL of mixed stop solution was added and the absorbance was read at 450 nm within 30 min.Serological tests for 2 tests were used to evaluate differences between genders in case and control groups. ANOVA was performed to assess any differences between ages in the different groups. Likelihood ratio tests were performed to assess interaction effects on the risk of gastric cancer by comparing the model that only involved the main effects and the full model that also contained the interaction term. A P-value <0.05 for all two-sided tests was regarded as statistically significant.All statistical analyses were carried out using SPSS 16.0 software . Pearson's χP = 0.018) in the smaller study population but no significant association was found in the further validation study of a larger population (unpublished data); PGC rs4711690, rs6458238, and rs9471643 polymorphisms were significantly associated with increased risk of atrophic gastritis  ERCC6 rs1917799 polymorphism was significantly associated with increased risk of gastric cancer  In our previous study, we found that pri-let-7a-1 rs10739971 polymorphism was significantly associated with increased risk of atrophic gastritis  and the result was statistically significant . As for the combination of two or more SNP-SNP interactions, the risk was often of moderate (OR≥1.5) or strong effect (OR≥2) ERCC6 single-locus showed a weak effect for gastric cancer risk (OR = 1.46) PGC single-locus demonstrated a weak protective effect for atrophic gastritis risk  ERCC6 rs1917799) was associated with gastric cancer risk, and the interactions of the other two pairs  were associated with atrophic gastritis risk. ERCC6 rs1917799 polymorphism and PGC rs6458238 and rs9471643 polymorphisms have been reported previously. In this study, we additionally analyzed pri-let-7a-1 rs10739971 polymorphism in the same group. The pair of pri-let-7a-1 rs10739971 polymorphism and ERCC6 rs1917799 polymorphism had an OR of their interaction of 2.59 for gastric cancer risk, which was greater than their individual single-locus effects of 0.92 and 1.07, respectively. Similarly, the pair of pri-let-7a-1 rs10739971 polymorphism and PGC rs6458238 polymorphism had an OR of their interaction of 2.77 for atrophic gastritis risk, which was larger than their individual single-locus effects of 0.74 and 1.23, respectively. The pair of pri-let-7a-1 rs10739971 polymorphism and PGC rs9471643 polymorphism had an OR of their interaction of 0.52 for atrophic gastritis risk. Interestingly, their individual single-locus effects were contrary to the above findings, with OR of 1.65 and 1.06, respectively, which deserves further independent replication of our findings.Among the SNPs from our group investigated during our candidate gene association study, PGC SNPs on atrophic gastritis risk, we further carried out interaction analysis of the three positive polymorphisms. The results showed that the OR for the risk of atrophic gastritis was 23.55 (P = 0.001). Generally, a single-locus has a weak effect (OR<1.5) on disease risk, and the combination of two or more SNP-SNP interactions often demonstrates a moderate (OR≥1.5) or a strong effect (OR≥2). In the present study, the OR of pri-let-7a-1 single locus was 1.61; the OR for interaction effects with ERCC6 and PGC were both more than 2; the combination of the three positive loci demonstrated an even higher OR of 23.55. These outcomes indicated the application prospects of a combination of two or more SNPs as potential biomarkers to predict disease risk.Because pri-let-7a-1 rs10739971 polymorphism demonstrated interaction effects with both PGC and ERCC6. Whether the SNP-SNP interaction effects observed in this study were because of binding of miRNA and its target genes still requires further functional experiments.Investigating the interaction effects of miRNA polymorphisms and gene polymorphisms will contribute to the comprehensive understanding of the gene network and the role of miRNA in the pathogenic process. Findings of SNP-SNP interactions often have underlying mechanisms rather than being merely statistical results This study had some limitations. First, although we had a relative large sample size of 471 gastric cancer patients, 645 atrophic gastritis patients and 717 controls, this sample size may still be inadequate for detecting interaction effects, particularly for rare alleles. Second, the mechanisms of the associated SNP-SNP interactions need to be clarified, and subsequent functional experiments are required. Third, several genome-wide association studies suggest an integration of data from genome-wide association studies of gastric cancer from Chinese populations; therefore, data should be integrated, and may be used as candidate polymorphism sites to be studied in the future. Fourth, the interaction between gene and environment for gastric cancer risk is an important factor to be considered, such as smoking and drinking. In this study, we collected some data on smoking and drinking, but there remains nearly a third of missing data from the samples. Interactions between genes and smoking and drinking behavior on gastric cancer risk should be examined.ERCC6 rs1917799 polymorphism might have an interaction effect on gastric cancer risk; and pri-let-7a-1 rs10739971 polymorphism might have an interaction effect with PGC rs6458238 and rs9471643 polymorphisms on atrophic gastritis risk. Some data have been collected providing for the possible construction of a gastric cancer network pathway. Future large-scale studies and mechanism experiments are required to confirm the findings of this study.This study, for the first time, reports that pri-let-7a-1 rs10739971 polymorphism and


CYBA C242T polymorphism is associated with coronary artery disease (CAD). Therefore, we undertook a comprehensive meta-analysis to more precisely evaluate the influence of this polymorphism on CAD and potential biases.Observational and experimental studies have thus far been unable to resolve whether the We screened MEDLINE, Embase, CNKI, Wanfang and CBM up to January 2013 and extracted data from 22 studies with 9,279 CAD patients and 9,349 controls. A random-effects model was exploited to synthesize the inconsistent outcomes of the individual studies, while addressing between-study heterogeneity and publication bias.CYBA C242T polymorphism conformed to Hard-Weinberg Equilibrium for all studies (P>0.05). Overall comparison of the T allele with the C allele produced a non-significant risk estimate for CAD but with striking heterogeneity . However, subgroup analysis by ethnicity documented that the T allele carriers had a marginal risk increase (21%) of CAD among Caucasians . Then data were divided into study design, the significance of CAD risk increase was substantially strengthened in matched case-control studies .Further meta-regression analysis identified that a large proportion of heterogeneity was explained by body mass index (BMI)  and study design .There was no obvious publication bias as verified by funnel plot and Egger's linear regression test .The CYBA C242T polymorphism might be a risk-conferring factor on developing CAD and BMI and study design were probable sources of between-study heterogeneity.Taken together, our results suggested the  Coronary artery disease (CAD) and myocardial infarction (MI) are principal public health contributors to worldwide death and disability, and as such are primary causes of economic burden CYBA gene which is located on 16q24. One of the most common polymorphisms of CYBA gene is C242T(rs4673), resulting in a non-conservative substitution of histidine for tyrosine at codon 72 and an alteration of NAD(P)H activity by disrupting the heme binding site NAD(P)H oxidase is the predominant cellular source of ROS in the context of atherosclerosis We collected data by searching from PubMed/MEDLINE, Embase, CNKI (China Nation Knowledge Infrastructure Platform), Wanfang and CBM  electronic databases up to January 2013. The search strategy followed the guidelines of the 2009 preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement  [17]. A All studies related to the association of the C242T polymorphism with CAD risk were potentially involved. The following criteria were adopted to the selection: (1) Articles published in English or Chinese journals or their supplements; (2) Studies providing adequate information on the C242T polymorphism by case-control status (studies without controls were excluded); (3) Published articles of human genetics (full texts or abstracts) without ethnicity restriction; (4) If articles involved more than one geographic or racial group, each group was considered as a separate study; (5) If multiple studies stemmed from the same population, only the largest scale study was included to avoid overlapping data; (6) The genotype frequency amongst control must conform to HWE.Standard definitions were used to diagnosis disease outcomes th and 95th percentiles).We undertook a standard data-collection protocol following the inclusion criteria described above. Two authors (Z.W. and Y. Lou.) independently extracted the relevant characteristics from each selected study and entered the data into separate databases in duplicate. Moreover, the results of data extraction were compared and any encountered divergences were resolved at a consensus meeting. The following information was gathered on the C242T polymorphism on the basis of different cohort: First author's name, publication year, ethnicity, country or area, study design, population source, disease outcomes, matching variables, clinical characteristics for case-patients and controls , the distribution of the C242T genotype both in patients and controls and conformity to genotype frequencies with HWE. Continuous variables manifested themselves as mean ± standard deviation (SD) or median  2 statistic, which determines the percentage of the between-study variability caused by heterogeneity rather than chance. Large values of I2 strongly implied the presence of between-study heterogeneity The synthetic studies' odd ratios (ORs) corresponding to 95% confidence intervals (CIs) for the relationship between the C242T polymorphism and CAD risk were calculated under different inheritance models, containing allele comparison (T versus C), dominant genetic model (CT+TT versus CC), recessive genetic model (TT versus CT+CC) and homozygote comparison (TT versus CC). We appraised the individual effect size together and modulated the study weights based on in-study variance by exploiting the DerSimonian & Laired method in a random-effects model. The possibility of heterogeneity was also assessed by using the Mantel-Haenszel model We undertook a sensitivity analysis by successive one-study removal approach to identify the most influential studies, likely introducing bias to the overall estimation http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl). Data processing and statistical analyses were performed using Review Manager software release 5.0  and Stata 11.0 . All P values were 2-sided.Consistency of the C242T polymorphism to HWE was examined via the chi-square test or Fisher's exact test based on a Web program ; these two studies were precluded. Because the study by Saha et al.et al.The flow diagram schematizing the selection process of included studies is shown in In total, 21 publications involving 22 studies (20 articles written in English HWE>0.05) are also listed in Our meta-analysis encompassed 22 studies with 9,279 CAD patients and 9,349 controls. The selected characteristics of these eligible studies were shown in 2 = 67.8%, Pheterogeneity<0.0001; dominant model: I2 = 65.7%, Pheterogeneity<0.0001; homozygote comparison: I2 = 35.7%, Pheterogeneity = 0.05), with the exception of the recessive model  . Because every subject contributes two observations under the allele comparison and we cannot a priori hypothesis that these two observations are independent, we computed log ORs with standard errors in each study and then used them in our meta-analysis. As a result, the risk estimates did not change if the clustering of the alleles in each subject was taken into account.Furthermore, we assessed the association of the C242T polymorphism with CAD risk under different genetic models. The summary results of the meta-analysis were shown in The striking heterogeneity identified across studies could influence the total outcome of the meta-analysis. Therefore, we performed a sensitivity analysis to look for the study or studies making the greatest contribution to heterogeneity. No single study or group of studies could be confirmed as disproportionately influencing the heterogeneity and ORs in total and subgroup analysis.We did not find any clear evidence to suggest that the first published study affected and brought about the ensuing replication via cumulative meta-analysis (data not shown). In addition, funnel plot analysis suggested that the overall results were not asymmetric and Egger's regression asymmetry test further verified an absence of publication bias  in the overall estimates .heterogeneity = 0.001, I2 = 75.6%; dominant model: P = 0.21, OR = 0.82, 95%CI 0.61–1.12, Pheterogeneity<0.0001, I2 = 77.4%) and in others group  without significance. Interestingly, the risk estimates of the C242T polymorphism with CAD differed between Caucasians and Asians, with an increased OR of CAD among the former . In particularly, we observed a marginal increased risk of CAD under the recessive genetic model  and in homozygote comparison  among Caucasians without distinct heterogeneity , Asian , and others (3 study recruited 435 cases and 378 controls). The ORs of the C242T polymorphism appeared to decrease in the Asian group  but not under the dominant model  in matched studies , this was not statistically significant. In further subgroup analysis, a lack of dramatic association of the C242T polymorphism with CAD risk was returned when using any genetic model in which data were categorized according to population sources and disease outcomes  and study design (P = 0.03) respectively, with CAD risk of the T allele being increasing in high BMI population  compared to low BMI population and in matched studies  compared to studies which did not mention whether the matched case-control study design was used . The adjusted Rwas used .2) was chosen as the median. Interestingly, most Caucasian studies (81.8%) had BMI≥25.8 kg/m2, while none of non-Caucasian studies had BMI≥25.8 kg/m2.Furthermore, we compared the descriptive factors between Caucasian and non-Caucasian studies to investigate underlying confounders which may influence the results .We found2 value significantly increased after adjustment for BMI.We took ethnicity and BMI into account by separately adding them into the multivariable regression. There was a decrease in the OR increase of population source, study design and BMI and a slight increase in OR increase of disease endpoint after adjustment for Caucasian/non-Caucasian, which was similar to that after adjustment for BMI. There were no substantial changes observed in the effect estimates of age, gender, HTN, DM and smoking status after adjusting for ethnicity and BMI. Nevertheless, the adjusted effects did not attain significance. The frequent reduction by adjusting may imply that confounding of the potential factors by ethnicity and BMI was likely to overestimate the influence of the study factors on the CAD risk estimates The correlation of the C242T polymorphism with CAD risk has yet to be clarified. Reckoning the CAD risk of the minor allele in independent case-control studies generated a conspicuous divergence, which was attributed to such causes as racial diversity, research strategy and limited sample size et al.et al.et al.et al.Given that distinct heterogeneity was present and could not be completely eliminated As well as the obvious association to ethnicity, other environment exposures have confounding potential for the interpretation of between-study heterogeneity. When taking multiple study-level covariates from clinical characteristics into account, we identified study design and BMI as exhibiting significance and positivity in univariate meta-regression analysis, and as such should be cautiously deciphered. Moreover, we found that there was a larger proportion of the Caucasian studies using matched study design compared to non-Caucasian studies. The studies among Caucasians were also more often performed in high BMI populations than those among non-Caucasians. Then accounting for the effects of ethnicity and BMI in the multivariable meta-regression, study design was no longer detected as a significant source of heterogeneity. Therefore, study design and BMI are likely confounders. The estimated advantage of Caucasians over non-Caucasians may be partially accounted for by the effect of study design and BMI and therefore, the effect of ethnicity on the variation cannot be regarded as an independent factor. Although meta-regression suggests an ecological correlation rather than a causal inference, our result implies underlying confounding of the environmental factors with genotype. Large amounts of observational studies have verified that increased BMI contributes causally to CAD CYBA gene and did not account for possible synergistic effects of other candidate genes or polymorphisms. Genetic susceptibility to CAD is most likely based on the interaction of polygenic and environmental factors Although the relatively large sample size and conformation to HWE strengthen our meta-analysis Experimental studies have suggested the C242T polymorphism affects the heme-binding site and alters electron flow and superoxide production Taken together, our meta-analysis, comprising 18,628 participants, implied that the T allele might carry an increased risk of CAD. However, it is conceivable that the C242T polymorphism has a heterogeneous effect on CAD across different ethnicities, being moderate among Caucasians but lacking significance among Asians. Our meta-analysis also highlights that it is necessary to consider potential gene-gene and gene-environment interactions when trying to interpret and combine observed data. For now,it remains unclear whether the T allele carriers have positive or negative effects on CAD and further population surveys and functional researches will be required to elucidate the true relationship.Checklist S1The PRISMA checklist for this meta-analysis.(DOC)Click here for additional data file.