Previously, TP53 codon 72 polymorphisms have been implicated as risk factors for various cancers. A number of studies have conducted on the association of TP53 codon 72 polymorphisms with susceptibility to breast carcinoma and have yielded inconclusive results. The aim of the present study was to derive a more precise estimation of the relationship.We conducted a search in the Medline, EMBASE, OVID, Sciencedirect, and Chinese National Knowledge Infrastructure (CNKI) without a language limitation, covering all papers published up to Jan 2009. The associated literature was acquired through deliberate searching and selected based on the established inclusion criteria for publications.A total of seventeen case-control studies, including 12226 cases and 10782 controls, met the included criteria and thus were selected. Ultimately, the relevant data were extracted and further analyzed using systematic meta-analyses. Overall, no associations of TP53 codon 72 polymorphisms with breast carcinoma were observed . In the subgroup analysis by ethnicity, statistically similar results were obtained when the data were stratified as Asians, Caucasians and Africans.TP53 codon 72 polymorphisms might not be a low-penetrant risk factor for developing breast carcinoma.Collectively, the results of the present study suggest that  Breast cancer is the second leading cause of cancer death in women, exceeded only by lung cancer in the world . It is bPreviously, a number of studies indicate that family history is a risk factor for breast cancer , indicatRecently, a growing body of research has conducted on the association of breast cancer risk with tumour suppressors. TP53, one of the most extensive studied genes as a tumor suppressor, has been thought to have a critical function in cell cycle regulation. In case of its mutation, this regulation could be lost, resulting in cell proliferation without control and development of cancer. Previously, TP53 mutation has been indicated to associate with risks of a number of cancers such as lung cancer, breast Recently, much attention has been focused on possible associations of TP53 polymorphisms and cancer risks. The most informative polymorphism in TP53 gene is located in exon 4 at codon 72, which encodes two distinct functional allelic forms arginine (Arg) and proline (Pro) because of a transversion G to C , resultiIn recent literature, inconclusive data regarding TP53 codon 72 were found in some cancers, such as gastric cancer in which controversial conclusions were obtained in Asians  and in iAs a single study may have been underpowered in clarifying the relationship of TP53 codon 72 polymorphisms with breast carcinoma susceptibility, in the present study we performed evidence-based quantitative meta-analyses that can increase statistical power to address the association.TP53, P53, codon 72, breast, carcinoma, neoplasm, tumor, cancer and polymorphism. The keywords were paired each time in order to get more relevant information. For example, the word "breast" was always kept and others were substituted in different moments.We carried out a search in the Medline, EMBASE, OVID, Sciencedirect, and Chinese National Knowledge Infrastructure (CNKI) without a language limitation, covering all papers published up to Jan 2009, with a combination of the following keywords: We evaluated potentially associated publications by checking their titles and abstracts and then procured the most relevant publications for a closer examination. Moreover, the reference lists of the selected papers were also screened for other potential articles that possibly have been missed in the initial search. The following criteria were used for the literature selection for the further meta-analysis:1. Studies concerning the association of TP53 codon 72 polymorphism with breast carcinoma;2. Case–control or cohort studies;3. Papers presenting the breast cancer diagnoses and the sources of cases and controls;4. Articles offering the size of the sample, odds ratios (ORs) and their 95% confidence intervals (CIs) or the information that can help infer the results;5. The number of individuals homozygous for arginine (Arg/Arg), proline (Pro/Pro) and heterozygous (Pro/Arg) in breast cancer cases and controls should be offered;6. The methods of data collection and analysis should be statistically acceptable.Accordingly, the following exclusion criteria were also used:1. The design and the definition of the experiments were obviously different from those of the selected papers.2. The source of cases and controls and other essential information were not offered;3. The genetic distribution of the control group was inconsistent with Hardy-Weinberg equilibrium (HWE).4. Reviews and duplicated publications.After searching, we reviewed all papers in accordance with the criteria defined above for further analysis.Data were carefully extracted from all eligible publications independently by two of the authors according to the inclusion criteria mentioned above. For conflicting evaluations, an agreement was reached following a discussion. If a consensus could not be reached, another author was consulted to resolve the dispute and then a final decision was made by the majority of the votes. The extracted information was entered into a database. For data not provided in the main text, the relevant information was obtained by contacting corresponding authors as possible as we could.P > 0.05, ORs were pooled according to the fixed-effect model (Mantel-Haenszel), Otherwise, the random-effect model (DerSimonian and laird) was used. The significance of the pooled ORs was determined by Z-test. The HWE was assessed via Fisher's exact test.The odds ratio (OR) of TP53 codon 72 polymorphisms and breast cancer risk was estimated for each study. The pooled ORs were performed for additive model (Arg/Arg vs Pro/Pro), dominant model (Arg/Arg+Arg/Pro versus Pro/Pro) and recessive model (Arg/Arg versus Arg/Pro+Pro/Pro), respectively. For detection of any possible sample size biases, the OR and its 95% confidence interval (CI) to each study was plotted against the number of participants respectively. A Chi-square based Q statistic test was performed to assess heterogeneity. If the result of the heterogeneity test was P = 0.05 (Nfs0.05) [Publication bias was assessed by visual inspection of funnel plots, in whicNfs0.05)  for the A total of 131 studies regarding TP53 codon 72 with respect to breast cancer were searched and screened for retrieval, of which 97 irrelevant studies were excluded. Then, 9 studies -34 were We established a database according to the extracted information from each article. The relevant information was listed in Table We analyzed the heterogeneity of Arg/Arg versus Pro/Pro and dominant model (Arg/Arg+Arg/Pro versus Pro/Pro) as well as recessive model (Arg/Arg versus Arg/Pro+Pro/Pro). As shown in Table Table In the present study, a total of 17 studies were included. Nevertheless, the study conducted by Weston et al.  concerneP > 0.05). For dominant model (Arg/Arg+Arg/Pro versus Pro/Pro), the data available for our meta-analysis were obtained from 18 case-control studies containing 12226 cases and 10782 controls, of which 11137 cases and 9444 controls had the combined genotypes of Arg/Arg and Arg/Pro, while 1089 cases and 1338 controls had the homozygote Pro/Pro genotype. The overall OR was 1.12 (95% CI = 0.96–1.32) and the test for overall effect Z value was 1.47 (P > 0.05). Similarly, for recessive model (Arg/Arg versus Arg/Pro+Pro/Pro), the data were extracted from the 18 case-control studies concerning 12226 cases and 10782 controls, of which 6288 cases and 5112 controls had the wild-type homozygote Arg/Arg genotype while 5938 cases and 5670 controls had the combined variant genotypes (Arg/Pro and Pro/Pro) of the TP53 codon 72. The overall OR was 1.13 (95% CI = 0.98–1.31) and the test for overall effect Z value was 1.65 (P > 0.05).As shown in Table Considering the possible impact of ethnic variation on the results, we conducted subgroup analysis concerning Asians, Caucasians and Africans, respectively. Likewise, the subgroup analyses failed to suggest marked association between TP53 codon 72 polymorphisms and breast cancer risk in Asians, Caucasians and Africans.In order to compare the difference and evaluate the sensitivity of the meta-analyses, we also presented the results of the fixed-effect models as listed in Table 0.05 for evaluation of the stability of the results. Consequently, the Nfs0.05 were 237, 143 and 271 for additive, dominant model and recessive model respectively, which were more than five times of the number of the included studies, suggesting that the results of these meta-analyses are relatively stable and the publication biases might not have an evident influence on the results of the meta-analyses.Funnel plots were created for assessment of possible publication biases. Then, Egger's linear regression tests were used to assess the symmetric of the plots. As shown in Table In the present study, the results of meta-analyses showed that individuals with TP53 codon 72 polymorphism might not have significant associations with increased or decreased susceptibility to breast carcinoma.A previous meta-analysis conducted by Koushik et al.  regardinThe underlying mechanisms by which TP53 polymorphism influences cancer risk are not fully understood. TP53 is the most frequently investigated gene that is often mutated in a variety of cancers. Nevertheless, several single-nucleotide polymorphisms have been studied and reported in TP53 gene . The polA well-known risk factor, HPV infection, is thought to have an association with increased susceptibility to some cancers such as cervical  and oralHeterogeneity is a potential problem when interpreting the results of meta-analysis . In the 0.05 showed no remarkable publication biases in the meta-analyses. Second, in the subgroup analysis, the number of studies regarding Africans was relatively limited. It may be underpowered to explore the real association. Thus, the results may be interpreted with caution. Third, whether the experimental and control groups were from the same socio-economic status or the same geographic area have not been clearly presented in some of the included original papers, leading to any possible biases. Furthermore, the sample sizes of some included studies are rather small, which might be one of the reasons contributing to the between-study heterogeneity. Therefore, a number of further studies with large sample sizes with well-matched controls are required. Besides, gene-gene and gene-environment interactions should also be considered in the further studies.Some limitations might be included in this study. First, in this meta-analysis, most published studies and papers written in English or Chinese were searched. Moreover, although papers written in some other languages, cited by PubMed, were also searched, it is possible that some related published or unpublished studies that might meet the inclusion criteria were missed. Hence, some inevitable publication biases might exist in the results, though the NfsIn summary, despite the limitations, the results of the present meta-analysis suggest that genetic variations of TP53 codon 72 may not have a marked association with breast cancer risk.The authors declare that they have no competing interests.WZ and YZ conceived of the study, and carried out the analysis of the literatures and drafted the manuscript. ZX carried out the collection of the literatures. LC helped with the statistical analysis and manuscript drafting. ZC and WZ conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


ALOX15, FADS2 and LPCAT2) and metabolites (lysolipids) were enriched in lipid metabolism pathways. Enzymes encoding for these genes are involved in membrane biogenesis and metabolism of fatty acids into pro-inflammatory and anti-inflammatory mediators. Correlation analysis indicated a strong negative correlation between ALOX15, FADS2, and IL5RA expression with 2-arachidonoylglycerophosphocholine levels in dual responders. However, measuring arachidonic acid and docosahexaenoic acid levels in a validation cohort of 19 participants indicated that the free form of DHA (nmoles/µg of protein) was significantly (p = 0.03) different between early and dual responders after allergen challenge. Collectively these results may suggest an imbalance in lipid metabolism which dictates pro- (anti-) inflammatory and pro-resolving mechanisms. Future studies with larger sample sizes may reveal novel mechanisms and therapeutic targets of the late phase asthmatic response.Some asthmatic individuals undergoing allergen inhalation challenge develop an isolated early response whereas others develop a dual response (early plus late response). In the present study we have used transcriptomics (microarrays) and metabolomics (mass spectrometry) of peripheral blood to identify molecular patterns that can discriminate allergen-induced isolated early from dual asthmatic responses. Peripheral blood was obtained prior to (pre-) and 2 hours post allergen inhalation challenge from 33 study participants. In an initial cohort of 14 participants, complete blood counts indicated significant differences in neutrophil and lymphocyte counts at pre-challenge between early and dual responders. At post-challenge, significant genes ( Asthma is the most common chronic lung disease, but remains poorly understood due to its complexity and heterogeneity The allergen inhalation challenge is a useful clinical model in order to study the mechanisms underlying asthmatic responses Recent advancements in ‘omics’ technologies such as transcriptomics and metabolomics have enabled the study of complex diseases such as asthma + cells after allergen inhalation challenge has been shown to differ between ERs and DRs Our laboratory has previously shown that significant changes in the whole blood transcriptome (mRNA and miRNA) of mild atopic asthmatic individuals can be identified two hours after allergen inhalation challenge 1 (forced expiratory volume in one second) 20  less than 16 mg/ml 2-agonists which were withheld 8 hours prior to spirometry measurements This study was approved by the Institutional Review Boards of the participating health research institutes . Following written informed consent, 33 individuals participated in the allergen inhalation challenge as part of the AllerGen Clinical Investigator Collaborative (CIC). All study participants had predicted FEV20] was calculated using the Cockcroft equation 1 of 20% was achieved in all participants. Study participants were classified as early responders (ERs) if the initial drop in FEV1 resolved back to baseline within 1 to 3 hours of allergen inhalation and if the maximum drop in FEV1 between 3 to 7 hours was less than 15%. Study participants were classified as dual responders (DRs) if in addition to the early response, the participants experienced a maximum drop in FEV1 of 15% or greater between 3 to 7 hours of allergen inhalation. Participants with a maximum late phase drop less than 15% but required a lower dose of methacholine [PC20] on the second MCT compared to the first MCT were also classified as DRs.A methacholine challenge test (MCT) was conducted one day prior and one day following allergen inhalation challenge. On day one, each participant underwent a MCT where methacholine [PCA standard operating protocol exists for all blood collection and processing at participating AllerGen CIC sites. Ten milliliters of venous blood was obtained from each subject, prior to challenge (pre) and 2 hours post-challenge. Leukocyte-enriched  and plasma fractions  were freshly prepared from the blood and frozen at −80°C, after which RNA was extracted using standard Qiagen kits. Complete blood counts and differentials  were obtained at the time of each blood draw . CBCs/differentials were obtained for all study participants in the discovery cohort.http://www.bioconductor.org/). All analyses were performed in the statistical computing program R 2.14.2 Whole transcript gene expression analysis of pre and post RNA samples (28 samples) of the discovery cohort was performed using Affymetrix Human Gene 1.0 ST Arrays at the Centre for Translational and Applied Genomics (CTAG) in Vancouver, British Columbia. All microarray data (CEL) files have been deposited in NCBI’s Gene Expression Omnibus (GEO Series GSE40240). The ‘affy’ package Metabolite profiling as well as informatics were performed by Metabolon Inc. . Samples from the discovery cohort were extracted and prepared for analysis using Metabolon’s standard solvent extraction method. The extracted samples were divided into equal parts for analysis on the GC-MS and LC-MS/MS platforms. Also included were several technical replicate samples created from a homogeneous pool, containing a small amount of all study samples. The Metabolon informatics system consisted of four major components, the Laboratory Information Management System (LIMS), the data extraction and peak-identification software, data processing tools for quality control and compound identification, and a collection of information interpretation and visualization tools. The hardware and software foundations for these informatics components were the LAN backbone, and a database server running Oracle 10.2.0.1 Enterprise Edition. General platform methods are described in the Supplementary Information.Arachidonic acid (AA) and docosahexaenoic acid (DHA) were profiled using plasma samples in a validation cohort of 19 study participants. Briefly, plasma was first suspended in 1 mM butylated hydroxyanisole (BHA) in chloroform and methanol (2∶1) in order to protect the integrity of the samples. Following a previously described method http://www.bioconductor.org/) and The Comprehensive R Archive Network (CRAN) (http://cran.r-project.org/).Moderated robust regression in the Linear Models for MicroArrays (limma) package Top biological functions and canonical pathways of differentially expressed genes were analyzed using Ingenuity Pathway Analysis (IPA). The filtering criteria included; organ: lung, and cells: immune cells. Gene networks consisting of hypothetical and proven relationships were also generated using IPA. Fisher’s Exact Test p-value of 0.05 and Benjamini Hochberg adjusted p-value (FDR) of 0.05 were used to determine significant canonical pathways and biological pathways.1) criteria; a drop of greater than 20% (1 in the DRs (21.3±3.2%) was approximately 4 times greater than that in ERs (5.1±1.4%).than 20% . During 9 cells) compared to DRs (2.6±0.5×109 cells). Complete blood counts with differentials were obtained from all samples in the discovery cohort. At pre-challenge, absolute neutrophil counts were significantly (p = 0.04) greater in ERs  resulted in 770 probe sets of which 497 annotated probe sets were retained for further analysis. From the same samples, the plasma was analyzed for 292 named and 201 unnamed metabolites; only the named metabolites were kept for downstream analyses.CXCR1), Nicotinamide phosphoribosyltransferase (NAMPT), C-C chemokine receptor type 7 (CCR7) and Lymphoid enhancer-binding factor 1 (LEF1) are abundantly expressed by neutrophils and lymphocytes. In fact the differences in gene expression between ERs and DRs are consistent with the differences in cell-type frequencies between ERs and DRs.  differentially expressed probe sets in DRs relative to ERs were identified at an FDR of 10%, after adjusting for age and sex. Ingenuity Pathway Analysis (IPA) indicated Connective Tissue Disorders, Immunological Disease, Inflammatory Disease and Skeletal and Muscular Disorder as the top biological functions at an FDR of 5% . Signifiand DRs. . TherefoEight metabolites were found differentially expressed (p<0.05) between ERs and DRs at pre-challenge. Plasma levels of six metabolites including bilirubin, 4-vinyl phenol sulphate, 2-arachidonoylglycerophosphocholine, methionine, N-acetylglycine and malate were significantly elevated in DRs compared to ERs. Methyl palmitate and mannose were significantly under-expressed in DRs relative to ERs.IL5RA), Fatty acid desaturase 2 (FADS2), Arachidonate 15-lipoxygenase (ALOX15), and Cold shock domain protein A (CSDA). FADS2 (Δ6-fatty acid desaturase) is a key enzyme in the biosynthesis of ω-6 and ω-3 fatty acids such as arachidonic acid (AA) and docosahexaenoic acid (DHA) LPCAT2), which is involved in membrane biogenesis 25 probe sets were differentially expressed post-challenge at an FDR of 10%, after adjusting for age and sex . Six of Eleven metabolites were differentially expressed (p<0.05) post-challenge . Four of25,14 (25 significant genes for 14 responders) and ΔM11,14 (11significant metabolites for 14 responders). 25,8 and ΔM11,8) and DRs  using a correlation cut-off of 0.5. RCCA indicated greater numbers of highly correlated (r>0.5) gene-metabolite clusters in dual responders (FADS2 and ALOX15) involved in lipid metabolism and IL5RA identified in Regularized Canonical Correlation Analysis (RCCA) was used to highlight clusters of correlated genes and metabolites. The ratio between pre and post gene expression levels (post/pre) was calculated for the 25 and 11 differentially expressed genes and metabolites for 14 responders resulting in 2 datasets; ΔGsponders  comparedsponders . InteresThese results may suggest that, the interaction of lipid metabolizing enzymes with membrane phospholipids in white blood cells may differ between early and dual responders. However, validation of the underlying mechanism is beyond the scope of this study. Instead we looked to see how general mediators of inflammation which are also components of cellular membranes such as arachidonic acid (pro- and anti-inflammatory) and docosahexasnoic acid (anti-inflammatory and pro-resolving) respond to allergen inhalation challenge.sn position 2 in AA or DHA. Lipid levels were compared between ERs and DRs at post-challenge  using a robust linear model adjusting for age and sex. At post-challenge, neither forms of AA were significant (Both free and phospholipid forms of arachidonic acid (AA) and docosahexaenoic acid (DHA) were measured in a validation cohort of 19 (8 ERs and 11 DRs) study participants challenged with various allergens including cat, grass and ragweed . The frenificant , whereasAsthma is a complex disease consisting of multiple sub-phenotypes such that current therapies perform well in some phenotypes compared to others Although many genes were differentially expressed between ERs and DRs prior to (pre-) allergen inhalation challenge, this may be influenced by significant differences in cellular frequencies between ERs and DRs . Figure FADS2 and ALOX15 as well as IL5RA, which may suggest that these expression differences may be originating from eosinophils. EMR1, an eosinophil-specific receptor was differentially expressed between ERs and DRs at post-challenge 2 α (cPLA2-α) induces up-regulation of arachidonic acid levels in eosinophils Differentially expressed genes at post-challenge were enriched for lipid metabolism and linoleic acid metabolism pathways . Figure Lipid metabolism was also enriched in the list of significant metabolites at post-challenge. Cortisol, an immunosuppressive and anti-inflammatory molecule decreased in ERs and increased in DRs after challenge. Cortisol bound to carrier proteins in the blood can be released at the site of inflammation by the catalytic activity of neutrophil elastase ALOX15) or produce (FADS2) arachidonic acid were negatively correlated with the arachidonic acid lysolipid (2-arachidonoylglycerophosphocholine) in DRs after challenge. Although this network is biologically plausible, experimental work to validate the interactions amongst these molecules in peripheral blood is beyond the scope of this study.Although each data set (transcriptomics and metabolomics) is useful in identifying differences between ERs and DRs, considering each data set independently may result in some loss of information as both data sets were obtained from the same individuals. Current bioinformatics tools contain poor annotations for many of the differentially expressed metabolites; therefore integration with the transcriptomic data was approached using statistical means. Correlation analysis identified interesting patterns between differentially expressed genes and metabolites in ERs and DRs at post-challenge. Interestingly, in the validation cohort of 19 study participants (8 ERs and 11 DRs), docosahexaenoic acid instead of arachidonic acid was differentially expressed between ERs and DRs at post-challenge. The free form of DHA was down-regulated in ERs post challenge compared to pre-challenge whereas little change occurred in DRs from pre- to post-challenge . It may To our knowledge, this study is the first of its kind to apply ‘omics’ methodologies to peripheral blood and identify molecular differences between two asthma phenotypes. These differences consist of factors associated with both systemic and airway inflammation. Although this study does not directly evaluate the impact of allergen inhalation on the airways, the downstream effects of this lung insult can provide significant evidence in understanding the molecular mechanisms underlying the early and late asthmatic responses. Our study has shown that careful selection of study participants is effective in detecting molecular differences between ERs and DRs. The use of multiple data sets from the same individuals can identify common biological functions and mechanisms and integration of these data sets may narrow down the number of generated hypotheses. Although many gene-metabolite clusters were identified, one biologically plausible gene-metabolite cluster was identified in DRs. Similar studies with larger sample sizes may reveal novel mechanisms, and targets for the pathogenesis and treatment of the late phase asthmatic response.Figure S1Top biological functions and canonical pathways at pre-challenge. The FDR, affy IDs and fold-change of genes were uploaded into IPA after which the following filters were applied: organ: lung, and cells: immune cells and FDR cut-off of 10%.(DOCX)Click here for additional data file.Figure S2Top biological functions and canonical pathways for differentially expressed (FDR = 10%) genes at post-challenge. (See (DOCX)Click here for additional data file.Table S1Complete blood counts and differentials. The Mean±SE of each cell count and differential at pre-challenge in early and dual responders. The Mean±SE of each cell count and differential at post-challenge  in early and dual responders.(DOCX)Click here for additional data file.Table S2Differentially expressed genes and metabolites at post-challenge . A. Differentially expressed genes at post-challenge (FDR<10%). B. Differentially expressed metabolites at post-challenge .(DOCX)Click here for additional data file.Table S3Differentially expressed lipids in the validation cohort. Levels (Mean±SE) of arachidonic acid and docosahexaenoic at post-challenge  in early and dual responders.(DOCX)Click here for additional data file.Table S4Named metabolites profiled in this study. Metabolites highlighted in red are differentially expressed at p<0.05.(PDF)Click here for additional data file.


We therefore aimed to meta-analyze three eNOS widely-evaluated polymorphisms, G894T (rs1799983) in exon 7, 4b/a in intron 4, and T−786C (rs2070744) in promoter region, in association with hypertension from both English and Chinese publications, while addressing between-study heterogeneity and publication bias.Numerous individually underpowered association studies have been conducted on endothelial nitric oxide synthase , and random-effects model was applied irrespective of between-study heterogeneity, which was evaluated by subgroup and meta-regression analyses. Publication bias was weighed using the Egger's test and funnel plot.There were total 19284/26003 cases/controls for G894T, and 6890/6858 for 4b/a, and 5346/6392 for T−786C polymorphism. Overall comparison of allele 894T with 894G in all study populations yielded a 16% increased risk for hypertension , and particularly a 32% increased risk  in Asians and a 40% increased risk  in Chinese. Further subgroup analyses suggested that published languages accounted for the heterogeneity for G894T polymorphism. The overall OR of allele 4a versus 4b was 1.29  in all study populations, and this estimate was potentiated in Asians . For T−786C, ethnicity-stratified analyses suggested a significantly increased risk for −786C allele  and −786CC genotype  in Whites. As an aside, the aforementioned risk estimates reached significance after Bonferroni correction. Finally, meta-regression analysis on other study-level covariates failed to provide any significance for all polymorphisms.eNOS G894T and 4b/a polymorphisms on hypertension in Asians, and T−786C polymorphism in Whites.We, via a comprehensive meta-analysis, ascertained the role of  The biological candidacy of eNOS in hypertension has been well-defined. For example, knockout mice deficient in eNOS developed hypertension as adults eNOS cDNA in mice reduced BP eNOS is highly polymorphic, it is of added interest to confirm which polymorphism(s) at eNOS might have functional potentials to affect the final bioavailability of eNOS, and thus the development of hypertension. A large panel of individually underpowered studies have been conducted on eNOS polymorphisms across different ethnic populations, yet with irreproducible and inconclusive results. To derive a more precise estimation, we therefore meta-analyzed three eNOS widely-evaluated polymorphisms, G894T (rs1799983) in exon 7, 4b/a (an insertion-deletion with 4a denoting four tandem 27-bp repeats and 4b five repeats) in intron 4, T−786C (rs2070744) in promoter region, in association with hypertension from the English and Chinese-published literature, while addressing between-study heterogeneity and publication bias.The viz. PubMed and Excerpta Medica Database (EMBASE), as well as two Chinese searching engines, viz. Wanfang database (http://www.wanfangdata.com.cn) and China Biological Medicine (CBM) (http://sinomed.imicams.ac.cn/index.jsp) as of Dec. 31, 2010. As a prerequisite, we restricted the search spectrum to articles written in English or Chinese language, and studies in human subjects. Boolean combinations of the key subjects (hypertension OR blood pressure) AND  AND  were used for identification. The full text of the retrieved articles was scrutinized to inspect whether data on the topic of interest were included. Also, reference lists of the retrieved articles and systematic reviews were checked to determine whether citations of articles that were not initially identified. If more than one geographical or ethnic populations were included in one report, each population was treated separately.Publications were identified from two international searching engines, eNOS G894T or 4b/a or T−786C polymorphism in association with hypertension; (ii) case-control or nested case-control or cross-sectional study; (iii) genotype/allele counts of G894T, 4b/a and T−786C polymorphisms between cases and controls for estimating odds ratio (OR) and 95% confidence interval (95% CI).Identified studies satisfied the following criteria: (i) evaluation of Hypertension was defined as systolic BP equal to or above 140 mmHg or diastolic BP equal to or above 90 mmHg or treatment with antihypertensive medication. Moreover, gestational hypertension studies were also included. However, studies evaluating secondary hypertension or other types of monogenic hypertension were excluded. If two or more studies shared the whole or part of study populations, the one with larger sample size was recruited.Two authors (W. Niu and Y. Qi) independently drew the following information from all qualified studies: first author's last name, publication date, population ethnicity, diagnosis criteria for BP, study design, baseline characteristics of the study population, and the genotype distribution in patients and controls. Any encountered discrepancies were adjudicated by a discussion until a consensus was reached.For consistency, continuous variables expressed as mean ± standard error (SE) were converted to mean ± standard deviation (SD). Moreover, the units of measures used in this study are transformed into the standard measurement units.eNOS G894T, 4b/a and T−786C polymorphisms with hypertension were calculated using the Stata software version 11.0 for Windows. In this meta-analysis, we implemented the random-effects model using the method of DerSimonian & Laird, instead of fixed-effects model, to bring the individual effect-size estimates together, and the estimate of heterogeneity was taken from the Mantel-Haenszel model Overall allelic/genotypic associations of I2 statistic, which was documented for the percentage of the observed between-study variability due to heterogeneity rather than chance with the ranges of 0 to 100%  Heterogeneity was assessed by the In addition, to look at more narrowly drawn subsets of the studies such as different study designs, separate analyses were undertaken in a sensitivity manner. Furthermore, to estimate the extent to which one or more covariates explain heterogeneity, meta-regression, as an extension to random-effects meta-analysis, was employed.Finally, publication bias was assessed using the Egger regression asymmetry test. The Egger's test detects funnel plot asymmetry by determining whether the intercept deviates significantly from zero in a regression of the standardized effect estimates against their precision.I2 test and Egger's test are recognized to have poor power, it is recommended that these statistics were statistically significant if the probability was less than 0.1 A naïve probability of less than 0.05 was judged significant, and Bonferroni correction was used to control for the multiple testing in view of three polymorphisms under investigation (significance was set at 0.05/3). Because the inconsistency index According to our search strategies, total 115 written-in-English (WIE) studies and 108 written-in-Chinese (WIC) studies were initially identified. Further application of our inclusion/exclusion criteria left 41 WIE (43 populations) I2 = 74.6%, P<0.0005) and high probability of publication bias as reflected by asymmetric funnel plot and significant Egger's test (P = 0.006). The magnitude of risk estimates for G894T genotype comparisons, as well as under dominant and recessive models were very similar to that in allele comparison (WIE = 0.702 and PWIC = 0.171) in each subgroup. Irrespective of published languages, we observed significant association of 894T with hypertension among populations of Asian descent  and among populations of Chinese descent  , even after Bonferroni correction, yet with strong evidence of heterogeneity  .In ethnicity-stratified analyses, no significance was reached in allele model for WIE studies among Asians , Arabs , Whites  and Blacks , whereas contrastingly, the odds of developing hypertension were remarkably potentiated, albeit suffering heterogeneity, in WIC studies, except for a Kazakh study .Additional stratification by study design showed slightly increased risk in hospital-based studies compared with population-based studies for both WIE and WIC studies, with significance reached in only WIC studies . After eTo investigate the possible sources of heterogeneity, we meta-regressed the genetic effects of G894T polymorphism on explanatory variables including averaged levels of age, gender , study design, BMI, TG, TC, and HDLC. Unfortunately, the confounders under study showed no statistical contribution to the prediction of hypertension in allele/genotype models for both WIE and WIC studies (data not shown).Egger = 0.289), genotype , dominant (PEgger = 0.213) and recessive (PEgger = 0.801) models in all qualified publications yielded low probability of publication bias. The significant overall estimate of allele 4a versus 4b was 1.29  for all studies, 1.21  for WIE studies and 1.47  for WIC studies, whereas there was strong evidence of between-study heterogeneity (P<0.005) (Considering the funnel plots  and statP<0.005) . CompariP<0.005) . Still, P<0.005) .Subgroup analyses were conducted to seek explanations for heterogeneity. Considering the study power, only those subgroups with ≥3 independent populations were reported. Comparison of allele 4a with 4b was significant, even after Bonferroni correction, in Asians , but not in Whites  . FurtherBesides the suggestive symmetry of funnel plots , Egger'sLikewise, stratification by study design showed no significance for all studies, as well as for WIE and WIC studies . HowevereNOS G894T and 4ab polymorphisms in Asians and T−786C polymorphism in Whites, the indications of between-study heterogeneity and publication bias, albeit disturbing, should merit serious consideration. To the best of our knowledge, this is the largest meta-analysis so far to investigate the association of eNOS three common polymorphisms with hypertension from the English and Chinese-published literature.Although our overall association produced significance for To avoid the disturbance of publication bias, we divided studies into characteristic-homogeneous groups such as the published language, and this situation has been greatly improved. For example, association of G894T polymorphism with hypertension showed high probability of publication bias in overall analyses, whereas none was observed in published language-stratified subgroups. At this point, we interestingly found remarkable heterogeneous associations by showing null association of 894T allele with hypertension in WIE studies, which was consistent with the results of two previous meta-analyses eNOS, two previous powerful meta-analyses indicated positive signals between eNOS variation and hypertension by consistently predisposing 4b allele to an increased risk, although publication bias obscured each study eNOS regulatory regions. On the other hand, 4b/a might interact with other polymorphisms to predisposing individuals to susceptibility or resistance to hypertension. As evidenced, Sandrim et al evaluated the eNOS haplotypes-based risk and demonstrated the haplotype −786C-4b-894G was linked to a protective effect on hypertension risk Although linkage information was lacking for eNOS T−786C polymorphism might not be a predisposing marker for hypertension. However, we extended this finding by showing significant association of T−786C in Whites. Factually, experimental studies have confirmed a functional role of this polymorphism on eNOS transcription activity by showing that the −786C-4b combination had the highest transcriptional activity As indicated by the results of previous meta-analyses To seek explanations for heterogeneity, besides subgroup analyses, an alternative approach is to perform a multivariate meta-analysis, in the form of a meta-regression, with the inclusion of covariates within this framework. This approach enables the moderating effect of a covariate, such as age or gender, to be tested formally. Unfortunately in this study, performing random-effects meta-regression analyses on various study-level covariates failed to provide any significant findings for all polymorphisms under study. However, it is important to bear in mind that meta-regression, although enabling covariates to be considered, does not have the methodological rigor of a properly designed study that is intended to test the effect of these covariates formally eNOS variation, some limitations merit serious consideration. First, all included studies had the cross-sectional design, which precludes further comments on cause-effect relationship eNOS polymorphisms, and did not evaluate other genes or polymorphisms. It is possible that the potential roles of G894T or 4b/a or T−786C polymorphisms are diluted or masked by other gene-gene or gene-environment interactions. Thus, the jury must refrain from drawing a conclusion until a large, well-performed Chinese study confirms or refuses our results.Despite the clear strengths of our study including large sample sizes and comprehensive evaluation of eNOS G894T and 4b/a polymorphisms on the development of hypertension for Asian populations and T−786C polymorphism for Whites. Although the publication bias was maximally avoided, presence of between-study heterogeneity could not be fully explained by our subgroup and meta-regression analyses. Although further analyses are warranted to investigate eNOS adjacent markers in a wider context, future studies should center on gene-gene and gene-environment interactions, as well as haplotype patterns.Taken together, via a comprehensive meta-analysis, we once again ascertained the role of Figure S1Allele comparison of eNOS G894T polymorphism for hypertension in Chinese (left pane) and Asians (right pane).(TIFF)Click here for additional data file.Figure S2Ethnicity-stratified allele comparison of eNOS G894T polymorphism for hypertension in WIE (left pane) and WIC (right pane) studies.(TIFF)Click here for additional data file.Figure S3Study-design-stratified allele comparison of eNOS G894T polymorphism for hypertension in WIE (left pane) and WIC (right pane) studies.(TIFF)Click here for additional data file.Figure S4Ethnicity-stratified comparison of eNOS 4b/a polymorphism for hypertension in all studies.(TIFF)Click here for additional data file.Figure S5Study-design-stratified allele comparison of eNOS 4b/a polymorphism for hypertension in all studies.(TIFF)Click here for additional data file.Figure S6Endpoint-stratified allele comparison of eNOS 4b/a polymorphism for hypertension in all studies.(TIFF)Click here for additional data file.Figure S7Study-design-stratified allele comparison of eNOS T−786C polymorphism for hypertension in all studies.(TIFF)Click here for additional data file.Table S1The baseline characteristics of all qualified studies in this meta-analysis.(XLS)Click here for additional data file.


Interleukin-1 beta (IL-1β), a pro-inflammatory cytokine, is emerging as a key mediator of carcinogenesis that characterizes host-environment interactions. Epidemiological studies investigating the association between two polymorphisms of IL-1B (−511C/T and +3954C/T) and cancer susceptibility have shown conflicting results. The aim of this study is to derive a more precise estimation of the relationship.Related studies were identified through a systematic literature search of PubMed and Web of Science from their inception to September 15, 2012. Summary odds ratios (ORs) and 95% confidence intervals (CIs) for the IL-1B −511C/T and +3954C/T polymorphisms and cancer risk were calculated. Heterogeneity among studies and publication bias were also tested.The meta-analysis included 91 case-control studies in 85 publications, 81 studies for the −511C/T (19547 cases and 23935 controls) and 26 studies for the +3954C/T polymorphisms (8083 cases and 9183). The pooled results indicated that IL-1B +3954C/T  was significantly associated with increased overall cancer risk, especially among hospital-based case-control studies . As for −511C/T, we observed an inverse relationship in cervical cancer  and hepatocellular carcinoma . Moreover, −511C/T was associated with risk of specific subtypes of gastric carcinoma.This meta-analysis suggested that both the IL-1B –511C/T and +3954C/T polymorphisms might modulate cancer susceptibility. Further well-designed studies based on larger sample sizes should be performed to confirm the findings. Cancer is considered to be a complex, multistep and fatal disease that results from interactions between environmental and genetic factors Interleukin-1 (IL-1) is a pro-inflammatory cytokine with multiple biological effects The IL-1B gene is highly polymorphic and base transitions between C and T at positions –511 , –31  and +3954  base pairs from the transcriptional site have been widely reported. In particular, the first two polymorphisms are located in the promoter region, and show high linkage disequilibrium It is not surprising that these functionally important polymorphisms in the IL-1B gene might be associated with cancer susceptibility. Recently, a meta-analysis of IL-1B –31T/C polymorphism and cancer risk has suggested that the –31C allele is a low-penetrance protective factor for the development of cancer Electronic databases (PubMed and Web of Science) were comprehensively searched using combinations of the terms “interleuk-1/IL-1” or “Interleuk-1B/IL-1B/IL-1 beta”, “polymorphism” and “cancer” or “tumor” . For each identified study, additional studies were sought from its citations, references and from the database option “Related Articles”.Eligible studies were selected according to the following explicit inclusion criteria: (i) case-control study evaluating the association between at least one of the two polymorphisms (IL-1B −511C/T and +3954C/T) and cancer susceptibility, (ii) sufficient genotype data presented to calculated the odds ratios (ORs) and 95% confidence intervals (CIs). Major reasons for exclusion of studies were: (i) only cancer group, (ii) no usable genotype frequency data, (iii) duplicate of earlier publication, (iv) publication not in English.Two investigators  extracted information from all eligible publications independently according to the criteria listed above. The following information was gathered from each study: the first author’s name, year of publication, cancer type, country of origin, ethnicity of subjects, source of control, genotyping method, number of cases and controls and genotype frequency . The quaP<0.05 was considered not compliant with HWE. The strength of the association between the two SNPs (IL-1B −511C/T and +3954C/T) and cancer risk was measured by ORs with 95% CIs. Pooled ORs were obtained from combination of single studies by homozygote comparison (TT vs. CC), heterozygote comparison (CT vs. CC), dominant model (TT+CT vs. CC) and recessive model (CC+CT vs. TT), respectively. The heterogeneity among different studies was checked by the Q-test P value is <0.10, a random effect model with the DerSimonian and Laird method was used to pool the results. Otherwise, a fixed-effect model with the Mantel-Haenszel method was then used We first calculated Hardy–Weinberg equilibrium (HWE) in the controls for each included study using a goodness-of-fit test and for which Sensitivity analysis was performed to assess the stability of the results by omitting a single study in this meta-analysis each time to reflect the influence of individual data on the pooled OR. To further explore the potential source of heterogeneity among studies and test the effects of study characteristics on the overall estimates, subgroup analyses and meta regression were performed stratified by cancer types , ethnicity , and source of controls .P<0.05 in Egger’s test indicated the presence of potential publication bias. All statistical analyses used STATA 12.0 . Except for heterogeneity statistics (where significance was declared if P<0.10), each P-value <0.05 was considered to be statistically significant. All P-values were 2-sided.Publication bias was diagnosed with Begg’s funnel plot and Egger’s linear regression method Detailed characteristics of the aggregated data for 91 case-control studies are summarized in Association of the IL-1B –511C/T polymorphism with cancer susceptibility: Analyses of 81 case-control studies including 19547 cases and 23935 controls were conducted to explore the relationship between −511C/T and cancer risk . However, there were no significant gene-environment interactions in this case.The genotype distribution of −511C/T among cases and infection-match controls was available in 20 studies that investigated gastric cancer infected by Association of the IL-1B +3954C/T polymorphism with cancer susceptibility: The analysis eventually included 26 case-control studies with 8083 cases and 9183 controls for IL-1B +3954C/T. P = 0.816), MAF (P = 0.050) and source of control (P = 0.308) only explained little of the heterogeneity.Since there was significant heterogeneity for IL-1B –511C/T in all genetic models, a univariable regression was conducted to explore the predefined possible source of heterogeneity . We idenP-value were 0.628, 0.788, 0.888 and 0.579 for homozygote comparison, heterozygote comparison, dominant model and recessive model, respectively  and their relationship to cancer susceptibility. Its strength was based on the accumulation of published data providing greater information to detect significant differences. Our results demonstrated that IL-1B +3954C/T was significantly associated with increased overall cancer risk, especially among hospital-based case-control studies. No significant association was observed between −511C/T and overall cancer risk. However, results from subgroup analyses indicated that −511C/T was associated with susceptibility of certain types of cancer. Given the critical roles of IL-1β in inflammation and carcinogenesis, it is possible that both IL-1B −511C/T and +3954C/T polymorphisms may modulate the risk of cancer development.Cumulative evidence suggests that IL-1β plays an important role in tumorigenesis and development. It is well known that IL-1 expression is elevated in most human cancers. High plasma IL-1β levels are associated with a significantly increased risk of cancer, and tumor patients with high IL-1 expression have worse prognosis than those without In the subgroup analysis according to cancer type, a significant association was detected between the -511T variant allele polymorphism and increased cervical cancer risk. Interestingly, this allele seemed to be a protective factor for the development of hepatocellular carcinoma. Some possible reasons may explain this discrepancy. First, the relatively small number of eligible studies in each subgroup might induce significant/insignificant association by chance due to insufficient statistical power. Second, there are some conflicting data regarding the effect of the IL-1B −511/−31 haplotype on gene expression. Ex vivo blood stimulation assay suggests that the -511T/−31C haplotype is significantly associated with an increase in LPS-induced IL-1β intracellular secretion Our results for gastric cancer were partly consistent with results from a previous meta-analysis performed by Xue et al. In the stratification analysis of source of control, significantly increased risk between the IL-1B +3954C/T polymorphism and cancer risk was detected among hospital-based studies, but not among population-based studies. This may be due to some selection biases existing in hospital-based studies because such controls might come from a population with a related disease and may not be a representative of the general population, especially when there is a relationship between the investigated genotypes and the disease conditions that hospital-based controls might have. Although it is more convenient to recruit hospital controls, to reduce biases in such genetic association studies it may be preferable to use population controls.Significant heterogeneity existed among studies for IL–511C/T in all comparisons, and meta regression indicated that MAF in different ethnicities partly accounts for the heterogeneity. Although we observed a wide variation of -511T allele frequency of control resources in different populations, and the MAF might reflect an environmental impact on gene distribution, subgroup analysis stratified by ethnicity did not detect any significant association between –511C/T and cancer risk in Asians nor in Caucasians. Cancer is a multi-factorial disease resulting from complex interactions between environmental and genetic factors Some limitations of our meta-analysis should be mentioned. Our results were based on unadjusted estimates because of the absence of available information. If more detailed individual data such as age, sex and exposure were available, a more precise analysis would be performed. Additionally, both the asymmetric funnel plot and Egger’s test indicated the existence of publication bias in two comparisons for the IL-1B +3954C/T polymorphism. This may be explained by the restriction of the meta-analysis to studies published in English. In spite of these limitations, our meta-analysis also had some advantages. First, it is the most comprehensive meta-analysis to evaluate the two polymorphisms (−511C/T and +3954C/T) and their relationship to cancer susceptibility. The number of cases and controls through the pooled studies could significantly increase the statistical power of the analysis. Second, all included studies had acceptable quality (scored at least 5).In conclusion, this meta-analysis indicated that IL-1B +3954C/T was associated with significantly increased overall cancer risk, especially among hospital-based case-control studies. The IL-1B −511T allele showed evidence of an association with increased cervical cancer risk but demonstrated a protective role in the development of hepatocellular carcinoma. Moreover, −511C/T was associated with risk of specific subtypes of gastric carcinoma. Further large-sample research should use standardized unbiased homogeneous cancer patients and well-matched controls to confirm our findings. Additionally, gene-environment interactions should be considered in future studies.Figure S1Minor allele frequencies of IL-1B −511C/T and +3954C/T polymorphisms among ethnicities of Asian, Caucasian and Others in controls.(TIF)Click here for additional data file.Table S1Main characteristics of the selected studies.(DOC)Click here for additional data file.Table S2Results of random effect meta-regression for search of source of heterogeneity for IL-1B –511C/T.(DOC)Click here for additional data file.Table S3Results of random effect meta-regression for search of source of heterogeneity for IL-1B +3954C/T in the dominant model.(DOC)Click here for additional data file.Table S4PRISMA Checklist for current meta-analysis.(DOC)Click here for additional data file.


Neural Tube Defects (NTDs) are among the most prevalent and most severe congenital malformations worldwide. Polymorphisms in key genes involving the folate pathway have been reported to be associated with the risk of NTDs. However, the results from these published studies are conflicting. We surveyed the literature (1996–2011) and performed a comprehensive meta-analysis to provide empirical evidence on the association.MTHFR C677T , MTHFR A1298C , MTR A2756G , MTRR A66G , and RFC-1 A80G . We found a convincing evidence of dominant effects of MTHFR C677T  and suggestive evidence of RFC-1 A80G . However, we found no significant effects of MTHFR A1298C, MTR A2756G, MTRR A66G in risk of NTDs in dominant, recessive or in allelic models.We investigated the effects of 5 genetic variants from 47 study populations, for a total of 85 case-control comparisons MTHFR C677T and a suggestive association of RFC-1 A80G with increased risk of NTDs. However, other variants involved in folate pathway do not demonstrate any evidence for a significant marginal association on susceptibility to NTDs.Our meta-analysis strongly suggested a significant association of the variant  Neural Tube Defects (NTDs) are congenital malformations of the brain and spinal cord in neurulation that occur between 21 and 28 days after conception Folic acid deficiency is relevant to the risk of the disease which was first demonstrated in seminal work done 36 years ago by Smithells et al. which showed that compared with the control group, women who had given birth to NTD children were significantly deficient in several micronutrients, especially folic acid, in diets and postpartum blood Emerging views of the evidence have begun to shed light on pathogenic mechanisms. One assumption is that folate transport may be affected by immunological responses and maternal autoantibodies that bind to the folate receptor can block the intracellular uptake of folate might cause NTDs –tetrahydrofolate (THF) and then it can accomplish the methylation cycle in order to function in folate metabolism. Inhibition in the folate metabolism pathway may induce a neural tube defect. Thus, the folate pathway genes that regulate the function of this cycle are widely investigated. Observations showed that some key genes involved in the methylation cycle of THF, such as the methylenetetrahydrofolate reductase (MTHFR), the reduced folate carrier (RFC) and the methionine synthase reductase (MTRR), combine with vitamin B12-dependent methionine synthase (MTR) function and transfer the methyl group to homocysteine to accomplish the circle MTHFR, A2756G in MTR, A66G in MTRR, A80G in RFC-1, have attracted most attention and may represent a substantial proportion of the risk of developing a neural tube defect as their key role in folate metabolism pathway Folic acid must first convert to its naturally bioactive formInterestingly, even though a number of studies investigated the correlation of the NTDs and the polymorphisms, no consensus has been reached. Some observations showed that folate pathway gene polymorphisms might be capable of inhibiting the folate pathway http://www.prisma-statement.org).To ensure the rigour of this current meta-analysis, we designed and reported it according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) MTHFR, MTRR, MTR and RFC associated with NTDs. The search strategy was based on combinations of the English and/or Chinese keywords, “MTHFR”, “MTRR”, “MTR”, “RFC”, “folate pathway” “polymorphism”, or “SNP” and “NTDs or Neural Tube Defects or spina bifida” without language restrictions. References of reviews and retrieved studies were also scanned.We searched PubMed, EMBase, ISI Web of Science, and Chinese Wan Fang Data databases for published articles from June 1996 to May 2011, which investigated at least one of the polymorphisms of MTHFR, MTRR, MTR and RFC; (3) presentation of data necessary for calculating odds ratios (ORs); (4) clear definition of NTDs. Animal studies, mother studies, reviews, and no specific data reported were excluded. Studies that duplicated other studies were eliminated, and only those whose design was complete were finally selected.The following inclusion criteria had to be fulfilled: (1) case-control study and cohort study design; (2) data on any, some or all polymorphisms in MTHFR, MTRR, MTR and RFC with NTDs.All the data were extracted independently by two reviewers (T. Zhang & R. Zhong). The following information was extracted from the eligible literature: year of publication, first author’s name, country, ethnicity, genotyping method, source of control, and matching variables of controls with cases. Counts of alleles or genotypes in both case and control groups were extracted or calculated from published data to re-calculate crude ORs and their 95% confidence intervals (95% CIs) for assessing the association of the polymorphisms in χ2-based Q statistic test was adopted to assess the case-between heterogeneity and, and heterogeneity was considered significant when P<0.05 for Q statistic or when I2 was above 75%. Data from the studies were combined in the Mantel-Haenszel Chi-square test by a random-effects model where heterogeneity was significantly present; otherwise, a fixed-effects model was applied. Pooled frequency analysis was performed in the method described by Thakkinstian P values are two-tailed with a significance level at 0.05. All statistical analyses were done using Stata Version 10. .Data from the case-control studies were summarized in two-by-two tables. In each table, crude ORs and their 95% confidence intervals (CIs) were calculated for each individual study based on the genotype data using the method as described by Zintzaras et al. MTHFR C677T, 22 studies of MTHFR A1298C, 8 studies of MTRR A66G, 9 studies of MTR A2756G and 4 studies of A80G. These studies enrolled 4374 cases and 7232 controls of MTHFR C677T, 2602 cases and 4070 controls of MTHFR A1298C, 703 cases and 1572 controls of MTRR A66G, 843 cases and 1006 controls of MTR A2756G and 1107 cases and 1585 controls of RFC-1 A80G.  and 34.2% (30.7%–37.8%); low in Asian healthy populations 20.7% (11.1%–30.3%). The frequency of risk C allele in A1298C also revealed differences–high in the Asian controls 42.8% (40.3–45.4) and low in Native American and European populations 19.6% (14.4%–24.7%) and 27.2% (24.1%–30.4%)  .p values for heterogeneity. A fixed-effects model was applied to the allelic and recessive models in the study of MTR A2756G and to the recessive model in the study of RFC-1 A80G, while a strict random-effects model was chosen for the rest of the studies in which the p values of heterogeneity(<0.05) showed significance. According to the method of choosing genetic model, we first calculated the ORs , and OR1≠OR2, so that we excluded the recessive model. Considering that OR3 was not significant and the heterogeneity was more significant in an allele model, a dominant model was finally determined. In the same way, we chose a dominant model for the remaining four SNPs. Among all the combined studies of SNPs and NTDs, MTHFR C677T showed an association with NTDs . There was no association observed between these four SNPs and NTDs and the results of other SNPs that we performed were negative. Specific ORs, I2 and p values are presented below. The results are presented in MTHFR C677T and A1298C was performed respectively. An empty regression was firstly run to estimate the baseline value for τ2 (τ12 = 0.098 and τ22 = 0.244), and then a series univariate model was conducted by adding single covariates including ethnicity of populations, source of controls, publication year and genotyping. In the univariate analysis, only the model including ethnicity and source of controls slightly reduced the τ2 value. Then we added the both two covariates, the τ12 value reduced to 0.089, R2 = 9.47% (1P = 0.103) and τ22 value reduced to 0.196, R2 = 22.52% (2P = 0.196), suggesting ethnicity and source of control cannot explain the major between-study heterogeneity. Studies of MTHFR C677T and A1298C were stratified to address heterogeneity while other SNPs were not stratified due to the importance priority and data availability. After stratification by sources of the controls, heterogeneity for MTHFR C677T was reduced in the substratification of the hospital based control group. In the population based control group, however, heterogeneity remained and the variant allele still conferred a significant increased risk. After stratification by ethnicity, heterogeneity in the Native American subgroup decreased with P = 0.139, I2 = 33.6%. Although heterogeneity remained, it should be noted that 22 studies of European subgroups also provided a significant correlation . For MTHFR A1298C, heterogeneity was reduced in the hospital based and Native American subgroups. Nonetheless, no significant association between MTHFR A1298C and NTDs was found  P = 0.162, I2 = 23.4), which indicated that this study may be the main origin of the heterogeneity. Nevertheless, our results did not change despite removing the data in this study. Relton’s study We performed a sensitivity meta-analysis to assess the effects of individual studies on pooled ORs. MTHFR C677T and NTDs was consistently observed with a narrowing of the 95% confidence interval through publication of the study in 2011. However, association study result of MTHFR A1298C and the risk for NTDs was negative.  MTHFR C677T and NTDs based on current limited knowledge of the exact mechanism.So far we have known that sufficient folate supplyment during the first four weeks of pregnancy can decrease the risk for NTDs by more than 50% MTHFR C677T shows a consistantly positive trend with objective facticity. Furthermore, sensitivity analyses have shown that none of the literature we included would influence the results negatively enough to reverse the results.In addition, the cumulative meta-analysis of 2 value of meta-regression reduced to 0.089, R2 = 9.47% (P = 0.103), suggesting ethnicity and source of control cannot explain the major between-study heterogeneity but it may be part of the origin of the heterogeneity. Further, we stratified all the studies into subgroups classified according to source of controls, ethnicity and genotyping. Reduced heterogeneity in Native America subgroup and hospital based subgroup was observed, and significant association was also observed among studies of Europe and Mixed populations. The result of the overall meta-analysis and cumulative meta-analysis for MTHFR A1298C did not support the MTHFR A1298C as an independent risk factor of NTDs. Considering that C677T and A1298C are both in MTHFR and are likely to interact. Also, lack of consensus in the results of individual studies as MTHFR C677T and A1298C may be due to the different environment background. The influence of dietary intake, especially folate intake, varied in different ethnic populations, which may well be relevant to the difference in prevalence and cannot be excluded in the this study. The between-study heterogeneity would also affect the results. However, the meta-regression analysis suggested that ethnicity and source of control may not be the major origin of the heterogeneity. In stratified analysis, we observed the heterogeneity reduced sharply only in Native American group and Taqman group. After checking all possible sources of errors, with the avalaible data we failed to exclude the influence of the between-study heterogeneity that existed in a relatively large meta-analysis. The heterogeneity might be due to many reasons, such as differences in maternal folate status and recruitment procedures of the study population.Nonetheless, the obvious evidence of between-study heterogeneity should be issued in our meta-analysis. The τMTR and mutations of the chaperone MTRR related to the activity of MTR may influence homocysteine levels resulting in severe disease phenotypes MTHFR, MTRR and MTR that are in folate metabolisms to analyse the association. However, the available evidence did not support MTRR A66G or MTR A2756G as an independent risk factor of NTDs. Some explanation might be responsible for the lack of correlation. First, the sample size of studies was relatively small, so that to detect a very small size effect may require much larger sample size. Second, the outcome of an NTD patient varies from livebirth to stillbirth as the severity varies in different subtypes. Thus, the effect of genetic variants on risk of NTDs may be underestimated if studies only collect livebirths and less severity cases. Third, potential gene-gene, gene-environment interaction may affect the current results.Under the hypothesis that loss-of-function mutations in RFC-1 A80G showed that the study of Relton (2004) RFC-1 A80G in fixed-effects model was 1.55(95%CI 1.24–1.92). Since the sample size was quite limited (901 cases and 983 controls), we must be cautious of the association and more studies should be required to add in to improve the precision of the result.The sensitivity analysis of MTHFR C677T alleles with NTD risk by performing a cumulative meta-analysis of 42 studies of MTHFR C677T that demonstrated results with a relatively narrow 95% confidence interval. We believe that as more studies are added to our meta-analysis, results would remain stable. Also, we observed a suggestive association beween RFC-1 A80G and the risk of NTDs. However we failed to find a correlation in the remaining SNPs.The current study strongly supports the association of Some limitations merit serious consideration in our meta analysis. Firstly, as the pressent meta-analysis was primarily based on unadjusted effect estimates, the confounding factors were not controlled. Additionally, with the eligible information and methodological limitations we cannot excluded the between-study heterogeneity that remained. Secondly, the effects of gene-gene and gene-environment interaction was not addressed in this study. Thirdly, we systematically searched a variaty of databases for published literature, however, we cannot excluded the possiblity of missing some.MTHFR C677T, and a suggestive association of RFC-1 A80G and the increased risk of NTDs while the other SNPs in our study failed to support an evidence of the association. As the low edge of 95%CI nearly touched the null value, our research should be viewed with caution. Further large and well-designed studies will be needed to clarify the association of the polymorphisms in the folate pathway genes.In summary, our studies demonstrated a significant correlation of Table S1Characteristics of studies on genetic polymorphisms in the folate pathway and Neural Tube Defects risk included in the meta-analysis.(DOC)Click here for additional data file.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.


MTHFD1 alleles, as well as the frequencies of MTHFD11958 genotypes  do not correlate with DS pregnancies, demonstrating no difference between the case and control groups, as opposed to the findings of Scala et al. (2006) on an Italian cohort.Recent reports linking Down syndrome (DS) to maternal polymorphism at the methylenetetrahydrofolate dehydrogenase (MTHFD) locus have generated a great interest among investigators in the field. In the current study, we examine one genetic polymorphism involved in homocysteine/folate pathway as a risk factor for DS in a Romanian urban–area women cohort. Our results show that the frequencies of  Down syndrome (DS) is a genetic disease resulting from the presence and expression of three copies of the genes located on chromosome 21. In 95% of DS cases, the nondisjunction of chromosome 21 occurs during meiosis I in the maturing oocyte; although advanced maternal age represents the major risk factor for DS, most children with this complex metabolic disease are born to young mothers (less than 35 years) [Abnormal folate metabolism and common folate–metabolizing enzyme variants have been described as possible risk factors for DS ,4. A defMethylenetetrahydrofolate dehydrogenase, MTHFD is an enzyme involved in folate metabolism, a trifunctional protein that mediates the interconversion of 5,10–MTHF, 5,10–methenylTHF, and 10–formylTHF. The last two are the donor cofactors for de novo purine and pyrimidine biosynthesis and, thus, for the biosynthesis of DNA .The MTHFD1 gene is located on chromosome 14 (14q24). A common MTHFD1 1958G>A polymorphism (Arg653Gln) reduces the enzyme's activity and stability and has been associated with an increased risk of several human diseases, including neural tube defects, congenital heart defects and unexplained second semester pregnancy loss ,12,12.Our paper addresses the relationship between MTHFD1 1958G>A polymorphism and the risk of DS pregnancy in a Romanian urban-area cohort of women.Our study included 26 women who gave birth to DS babies and 46 control mothers of healthy children. The case mothers were interviewed and a questionnaire regarding age, place of residency  during the pregnancy period, education, obstetric history, and periconceptional folic acid intake was completed. The DS mothers were younger than 40 years old and 7 of them had a history of spontaneous miscarriages (26.92%). In all cases, their babies were confirmed of having classic trisomy 21. The control blood samples were collected from 46 women who gave birth only to healthy children, without any history of miscarriages or abnormal pregnancies. An informed consent was obtained from all participants. All women in our study reside in the same geographic area and have a similar social background. Peripheral blood samples (5 ml) were collected on EDTA from all participants. Genomic DNA was isolated from the whole blood by using PeqGOLD Blood DNA mini kit, while following the manufacturer's instructions. The presence of MTHFD1 1958G>A mutation was analyzed by polymerase chain reaction followed by a restriction fragment length polymorphism analysis–allele specific restriction digestion with MspI (PCR–RFLP). Primers for amplification were forward: 5'–CCTGGTTTCCACAGGGCACTC–3' and reverse 5'–CCACGTGGGGGCAGAG GCCGGAATACCGG–3'. PCR conditions were denaturation 94 degrees C (1 minute), annealing 60 degrees C (1 minute), elongation 72 degrees C (1minute) for a total of 36 cycles. The PCR amplification generated a 310 bp fragment. The transition observed in MTHFD1 1958G>A variant abolishes a MspI restriction site; therefore after MspI digestion of the PCR product, the A–allele was detected by the presence of 282 and 28 bp fragments, while the G–allele was identified by the presence of 196, 86 and 28 bp fragments.Allele frequencies were calculated for each genotype, and the differences in allele frequencies between mothers of children with DS and control mothers were determined by using a chi–square test. Expected genotype frequencies were calculated from the allele frequencies under the assumption of Hardy–Weinberg equilibrium. The interaction between the two MTHFD1 genotypes was evaluated by calculating the odds ratios and p values for mutant genotypes. The analyses were performed while using the SPSS softwareIn this study, we examined one polymorphism in methylenetetrahydrofolate dehydrogenase (MTHFD1)–gene encoding a folate–metabolizing enzyme–as a maternal risk factor for meiotic nondisjunction of chromosomes 21, causing DS, in a cohort of Romanian mothers. The investigation of the polymorphism was addressed by PCR amplification of genomic DNA by using the primers described in Materials and Methods section, followed by restriction digestion with an appropriate endonuclease. The results of the mutational analysis are shown for a few representative cases in the .As observed in 2: 0.22, p value 0.63).The allele frequencies of MTHFD1 1958G>A in DS mothers and control mothers are listed in As far as genotypic frequencies are concerned, GA heterozygous genotype frequencies of MTHFD1 at position 1958 were higher among DS mothers than among controls mothers  0.54–5.26) indicating that this polymorphism may have a genetic impact upon the risk of DS. AA homozygous genotype frequency at this position was higher in control mothers than in DS mothers , although not at a statistically significant level. Several studies performed in the last decade suggest a possible contribution of an impaired folate metabolism to MTHFD1 DS risk, and the current opinion is that the presence of mutant alleles in the genome might affect the risk of the disease. However, the studies performed so far, have often provided conflicting results ,14,15,16Scala et al. investigated the possible contribution of the MTHFD1 1958G>A polymorphism as a maternal risk factor for having a DS child and observed positive interactions for the combined MTHFD1 1958AA/RFC1 80GG genotype . We inveWe did not find any statistically significant association between MTHFD1 polymorphic genotype and the history of DS pregnancies; thus, the relationship between MTHFD1 polymorphism and DS appears to be only a supposition and the next step in our study is the catamnestic evaluation of our patients with DS babies for two years. Additional studies are essential to unravel the complex relationship between genes, micronutrients, and folate/methyl metabolism.


The objective of this study was to explore whether the Fas rs180082 polymorphism confers susceptibility to cervical cancer.The Fas rs180082 polymorphism and cervical cancer risk was assessed by odds ratios (ORs) together with their 95% confidence intervals (CIs).The relevant studies were identified through a search of PubMed, Excerpta Medica Database (Embase), Elsevier Science Direct and Chinese Biomedical Literature Database (CBM) until July 2012. The association between the Fas rs180082 polymorphism and cervical cancer susceptibility in overall population . In subgroup analysis, similar results were found in Asian  and African .A total of 7 case–control studies were eventually identified. We found no association between Fas rs180082 polymorphisms with cervical cancer susceptibility. However, larger scale primary studies with the consideration of gene–gene and gene–environment interactions are still required to further evaluate the interaction of Fas rs180082 polymorphism with cervical cancer susceptibility.This meta-analysis has shown that there is a lack of association of the Fas  In recent years, cervical cancer has been the second frequent women malignancy worldwide, with an estimated global incidence of 493,243 new cases and 273,505 deaths per year . TherefoIt is widely accepted that specific human papillomavirus (HPV) types are the central etiologic agent of cervical carcinogenesis. Other environmental and host factors also play important roles in the persistence of HPV infection and further malignant conversion of cervical epithelium . A numbers180082 and FasL rs763110. In recent years, these polymorphisms have attracted widespread attention. However, the majority of studies focused on Fas rs180082 polymorphism.One of the most important mechanisms for HPV infection control of human immune system is to induce apoptosis of HPV-infected cells . Apoptosrs180082 polymorphism and cervical cancer risk has been observed in previous studies. Although a lot of studies had shown the possible involvement of Fas rs180082 polymorphism in the pathogenesis of cervical cancer, the results were not always consistent. Most studies showed that there was a lack of association of Fas rs180082 polymorphism with cervical cancer risk [rs180082 polymorphism and cervical cancer risk, we performed a meta-analysis of all eligible case–control studies in this article.In particular, the important polymorphism of Fas, the A/G mutation at position -670 loci, has been intensively taken into account. Furthermore, the relationship between Fas cer risk -15. Howecer risk . Furthercer risk . Therefors180082 polymorphism and cervical cancer susceptibility. The search strategy was based on combinations using the Mesh terms as follows: (“Fas”) in combination with (“polymorphism” or “polymorphisms” or “variant” or “SNP” or “mutation”) in combination with  for all publications on the association between Fas rs180082 polymorphism and cervical cancer susceptibility. No language or country restrictions were applied. All eligible studies were retrieved, and their bibliographies were checked for other relevant publications. Review articles were also inspected to find additional eligible studies. For the use of these clinical materials for research purposes, prior consents from the patients and approval from the Ethics Committees of the First Affiliated Hospital of Guangxi Medical University were obtained.We searched electronic databases PubMed, Excerpta Medica Database (Embase), Elsevier Science Direct and Chinese Biomedical Literature Database (CBM) updated on July 2012 for all publications on the association between Fas rs180082 polymorphisms and cervical cancer risk; (c) the papers must provide sufficient data including distribution of genotype and allele frequency; (d) when multiple publications reported on the same or overlapping data, we chose the most recent or largest population. Studies were excluded if one of the following existed: (a) studies that contained overlapping data; (b) Not offering the source of cases and controls or other essential information; (c) studies in which family members had been studied because their analysis are based on linkage considerations; (d) Reviews and repeated literature were also excluded.Studies included in our meta analysis had to meet the following inclusion criteria: (a) a case–control study; (b) information on the evaluation of Fas Two investigators (Xu Chen and Wuning Mo) independently extracted the information with the standard protocol and the result was reviewed by a third investigator (Qiliu Peng).From each study, the information we extracted the name of first author, year of publication, country of origin, ethnicity of the population studied, the number of cases and controls, allele frequency, definition of cases, and genotype distribution in cases. Different ethnic descents were categorized as White, Asian, or African. For studies including subjects of different ethnic groups, data were extracted for ethnic group whenever possible.2 = 100% * (Q–df)/Q [Data were analyzed mainly using the STATA Software . For each study, odds ratio (OR) and its 95% confidence interval (95% CI) were calculated to assess the association strength. Meta-analysis was performed for the polymorphisms that had been investigated in at least two studies. We examined the relationship between the allele, as well as genotypes and susceptibility to cervical cancer. The heterogeneity between the studies was assessed by the χ2-test based Q-statistic. A significant Q-statistic (P < 0.10) indicated heterogeneity among the studies, and so the summary OR estimate of each study was calculated by the random -effects model (DerSimonian and Laird method) . Otherwi(Q–df)/Q . The I s(Q–df)/Q . The ovePublication bias was observed with the funnel plot  using thrs180082 polymorphism on cervical cancer susceptibility. Eleven of these articles were excluded: one of these articles was a review [Based on the search criteria, the literature search identified 17 related articles relevant to the role of the Fas a review , one wasa review , one wasa review , one dida review , two foca review ,28, two a review ,30, and a review -33. Manua review -16. A fla review  literatua review -14,16, 2a review  and one a review . The gena review ,13.rs180082 polymorphism and cervical cancer risk are shown in Table Summary results of this meta-analysis for the association between Fas Although 2 studies ,13 did nThere was significant heterogeneity for heterozygote comparison of dominant model comparison (GA+AA vs. GG: P = 0.007) and recessive model comparison (GG vs. GA+AA: P= 0.001). Then, we assessed the source of heterogeneity for heterozygote comparison (GA+AA vs. GG) by ethnicity. As a result, ethnicity  was found to contribute to substantial heterogeneity.Begg funnel plot was created to assess the publication bias of literatures. As shown in Figure ′ upstream promoter region of the gene. SNP at -670 in the enhancer region (A/G) locates at a binding element of gamma interferon activation signal (GAS). Homozygous for G allele (GG) could lead to a complete deletion of the binding sequence of transcription element GAS, which is responsible for the signal emanated through STAT1, and in a significant alteration in the gene expression [The alterations of apoptosis-related genes are probably contributing to malignant tumors, including cervical cancer . Fas is pression .rs180082 polymorphism and cervical cancer risk, its eligible studies are not quite comprehensive [rs180082 polymorphism and cervical cancer risk. This update meta-analysis includes seven latest case–control studies, five of which are published in recent 5 years. Despite the number of eligible studies in this meta-analysis is still infinite, all the studies included in this meta-analysis, which are based on stricter search criteria, are more reliable and lead to a more accurate result.Although previous meta-analysis maybe involves some parts of the relationship between Fas ehensive . First, ehensive ,33. Howers180082 polymorphism were contradictory. These inconsistent results were possibly due to small effect of the polymorphism on cervical cancer risk or the relatively low statistical power of the published studies. Consequently, the meta-analysis was needed to provide a quantitative approach for combining the results of various studies with the same topic and for estimating and explaining their diversity.As for cervical cancer risk, the previous results of the studies involving Fas rs180082 polymorphism and cancer risk is limited. Some studies before had shown that there was an association between Fas rs180082 and some cancers, including nasopharyngeal carcinoma [So far, the number of the studies that focus on the relationship between Fas arcinoma , lung caarcinoma ,38, gastarcinoma  and leukarcinoma . Otherwiarcinoma .rs180082 polymorphism and cervical cancer susceptibility. However, Ueda, et al. found that polymorphism of Fas gene promoter -670 may be associated with the risk of cervical cancer in a Japanese population. It should be pointed out that the study which focused on Japanese population is based on small sample size (<200 subjects), so that we should be cautious to the result of this study. Consequently, our data failed to find a relationship between Fas rs180082 polymorphism and cervical cancer risk. The present meta-analysis, which included 1856 cases of cervical cancer and 2097 controls, suggested that there was no association between Fas rs180082 polymorphism and cervical cancer susceptibility.The studies included in our meta-analysis may differ in some aspects. Koushik, et al. conducted the first African population study with 447 women with invasive cervical cancer and 424 healthy women controls . Li, et rs180082 polymorphism on cervical cancer susceptibility. However, the conclusion should be cautious. Because the sample size of studies included in our meta-analysis is relatively small, especially in the African population. Our results may be underpowered so that further studies need to be conducted to increase the statistical power. The genetic distance between different ethnicities considered together could be substantial. It is not excluded that true effects are present in one specific sub-population, but undetected due to lack of statistical power or diluted/cancelled out by population stratification issues when different study populations are grouped together for analysis.Considering the results may be due to different ethnicity, we further conducted subgroup analysis according to ethnicity. When stratifying for ethnicity, significant association was detected in neither Asian populations nor African populations, suggesting that the genetic background or environment they live in may not influence the Fas Some limitations of this meta-analysis we need to pay attention to. First, our results are based on unadjusted estimates and a more precise analysis stratified by age, different lifestyle related habits and different grades of cervical cancer could be performed if individual data were available. Second, lack of the original data of the reviewed studies limited our precise estimation of the relation, which might cause some bias. Third, the lack of original study limited our further evaluation of potential interactions because the interactions between gene-environment interactions may modulate cancer risk.rs180082 polymorphism with cervical cancer risk is statistically more powerful than any single study. Second, the studies retrieved were the latest, half of which were published in the recent 3 years. Third, the quality of case–control studies included in our Meta analysis was satisfactory and met our inclusion criteria.Despite these limitations or disadvantages, our meta-analysis still had some advantages. First, a systematic review of the association of Fas rs180082 polymorphisms with cervical cancer. However, larger scale primary studies with the consideration of gene–gene and gene–environment interactions are still required to further evaluate the interaction of Fas rs180082 polymorphism with cervical cancer susceptibility.In summary, the present meta-analysis provides information that there is a lack of association of the Fas In the past five years we did not receive any reimbursements, fees, funding, or salary from an organization that may in any way gain or lose financially from the publication of this manuscript. We do not hold any stocks or shares in an organization that may in any way gain or lose financially from the publication of this manuscript. We do not hold and we are not currently applying for any patents relating to the content of the manuscript. We do not receive reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript. There are no any non-financial competing interests  to declare in relation to this manuscript.XC collected the literature data, developed the statistical model, carried out the software implementation and drafted the manuscript. QP collected the literature data, read the full text articles, and checked the model and results. XS collected the literature data and read the full text articles. WM helped with the discussion both in the theoretical development and English copyediting. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/14/71/prepub


The mean (sd) MN frequency of women with FM (51.4 (21.9)) was significantly higher than that of controls (15.8 (8.5)) . Significant differences (n = 69 sites) in methylation patterns were observed between cases and controls considering a 5% false discovery rate. The majority of differentially methylated (DM) sites (91%) were attributable to increased values in the women with FM. The DM sites included significant biological clusters involved in neuron differentiation/nervous system development, skeletal/organ system development, and chromatin compaction. Genes associated with DM sites whose function has particular relevance to FM included BDNF, NAT15, HDAC4, PRKCA, RTN1, and PRKG1. Results support the need for future research to further examine the potential role of epigenetic and acquired chromosomal alterations as a possible biological mechanism underlying FM.Fibromyalgia (FM), characterized by chronic widespread pain, fatigue, and cognitive/mood disturbances, leads to reduced workplace productivity and increased healthcare expenses. To determine if acquired epigenetic/genetic changes are associated with FM, we compared the frequency of spontaneously occurring micronuclei (MN) and genome-wide methylation patterns in women with FM ( To adjust for the observation that β values from the Infinium II designed probes have a compressed range compared to the β values from the Infinium I design [β values for the Infinium II designed probes [Because the 450 K HumanMethylation assay includes both the Infinium I design (which includes two probes for interrogating a CpG site) and the Infinium II design (which includes only one probe), the GC content was plotted separately by Infinium design type for the fully methylated sample, for which all CpG sites are expected to have consistently high β values . Based oβ values . Becauseormality . Prior tI design , 45, 46,β values from the patient and control samples. For each CpG site, differential methylation between specimens collected from women with FM and controls was tested using the moderated t-test in the limma Bioconductor package [P values were used to estimate the false discovery rate (FDR) following Benjamini and Hochberg's [Statistical analyses were then performed on the peak corrected logit transformed  package , 52 in t package . To adjuchberg's  method. chberg's  was usedr = 0.717; P = 0.021) and the healthy controls   (P = 0.997), smoking history , or alcohol use . To assess if there might be a cumulative biological effect associated with experiencing symptoms associated with FM, we compared MN frequencies for the case subjects (n = 10) with the total number of years that had lapsed since these women received their diagnosis of FM. While there was a trend toward a positive correlation between a woman's MN frequency and the number of years since she was diagnosed with FM (ranged from 2 to 19 years), this relationship did not reach significance in this small pilot study (P = 0.134)  . After c× 10−11) . The inc= 0.134) .After completion of the quality control assessments that were performed to remove any potential biases associated with probe sequence length, probe GC content, and inclusion of SNPs , a total3.26-fold higher than the level seen in the healthy controls. In comparison, patients who have cancer have been noted to have 1.37- to 3.13-fold higher frequencies of MN when compared to healthy controls [While the sample size in this pilot study is small, the MN frequency patterns of both the case and control women showed an age-related increase, which is a finding that is in agreement with the age-related increase that has consistently been reported in larger studies , 41. Intcontrols , 58. Givcontrols ; patientcontrols ; and aducontrols . The precontrols . Given tcontrols  and envicontrols , 41. Envcontrols , 63, horcontrols , and expcontrols . One cancontrols . In turnWhile limited in number, studies performed to assess the effect of methylation on chromosome segregation have consistently shown an increase in the frequency of cytogenic abnormalities associated with perturbations in the methylation status of chromosomes . In thisThe results of genome-wide methylation studies have provided insight regarding the role of genes and environmental influences for a variety of conditions, with many of these investigations focusing on the areas of cancer and psychiatric conditions , 75. HowIn summary, the results of this pilot study suggest that chromosomal instability and alterations in methylation are present in women with FM. If these results can be confirmed, they could provide a basis for improving our understanding of the biological changes leading to the development of FM and may provide a basis for stratifying patients based on their epigenomic and symptom patterns. Moreover, since epigenetic changes demonstrate plasticity , the rec