In this study, Mendelian randomization was used to study genes known to modify iron levels, and the effect of iron on Parkinson's disease (PD) risk was estimated. Based on estimates of the genetic effects on both iron and PD obtained from the largest sample meta-analyzed to date, the findings suggest that increased iron levels in the blood are associated with a 3% reduction in the risk of Parkinson's disease for every 10 µg/dL increase in iron. The results of this analysis have potentially important implications for future research into the prevention of Parkinson's disease.Please see later in the article for the Editors' Summary Although levels of iron are known to be increased in the brains of patients with Parkinson disease (PD), epidemiological evidence on a possible effect of iron blood levels on PD risk is inconclusive, with effects reported in opposite directions. Epidemiological studies suffer from problems of confounding and reverse causation, and mendelian randomization (MR) represents an alternative approach to provide unconfounded estimates of the effects of biomarkers on disease. We performed a MR study where genes known to modify iron levels were used as instruments to estimate the effect of iron on PD risk, based on estimates of the genetic effects on both iron and PD obtained from the largest sample meta-analyzed to date.HFE rs1800562, HFE rs1799945, and TMPRSS6 rs855791. Estimates of their effect on serum iron were based on a recent genome-wide meta-analysis of 21,567 individuals, while estimates of their effect on PD risk were obtained through meta-analysis of genome-wide and candidate gene studies with 20,809 PD cases and 88,892 controls. Separate MR estimates of the effect of iron on PD were obtained for each variant and pooled by meta-analysis. We investigated heterogeneity across the three estimates as an indication of possible pleiotropy and found no evidence of it. The combined MR estimate showed a statistically significant protective effect of iron, with a relative risk reduction for PD of 3%  per 10 µg/dl increase in serum iron.We used as instrumental variables three genetic variants influencing iron levels, Our study suggests that increased iron levels are causally associated with a decreased risk of developing PD. Further studies are needed to understand the pathophysiological mechanism of action of serum iron on PD risk before recommendations can be made.Please see later in the article for the Editors' Summary Parkinson disease is a degenerative disorder of the central nervous system caused by the death of dopamine-generating cells in the substania nigra, a region of the midbrain. The earliest symptoms are usually movement-related and include tremor, slow movements, and difficulty walking, and later cognitive and behavioral problems may arise, with dementia commonly occurring in the advanced stages of the disease. Parkinson disease affects around ten million people world-wide and incidence increases with age, with men more affected than women. To date, the causes of Parkinson disease remain unknown although a combination of genetic and environmental factors is thought to play a role. Identifying possible modifiable risks is an important step in the possible prevention of Parkinson disease.Previous studies have shown a possible association between lower blood levels of iron in people with Parkinson disease compared with controls, although the quality of these studies makes this finding difficult to interpret. So in this study, the researchers used a mendelian randomization approach to investigate whether there was any evidence of an effect of blood iron levels on the risk of Parkinson disease and if so to further explore the direction and scale of any link. Mendelian randomization is a method of using measured variation in genes of known function to examine the causal effect of a modifiable exposure on disease in situations where it is inappropriate to perform a randomized controlled trial.HFE and TMPRSS6. For each polymorphism, they performed a meta-analysis combining the results of studies investigating the genetic effect on iron levels, which included almost 22,000 people from Europe and Australia, and a meta-analysis of studies investigating the genetic effect on the risk of Parkinson disease, which included a total of 20,809 people with Parkinson disease and 88,892 controls from Europe and North America. They then performed three separate mendelian randomization analyses to estimate the effect of iron on Parkinson disease for the three polymorphisms. By combining the three estimates, they obtained a statistically significant odds ratio of 0.97 for Parkinson disease per 10 µg/dl increase in iron, corresponding to a 3% reduction in the risk of Parkinson disease for every 10 µg/dl increase in blood iron. Since genotype influences on blood iron levels represent differences that generally persist throughout adult life, the combined mendelian randomization estimate reflects an effect of iron over the course of a lifetime.The researchers estimated the effect of blood iron levels on the risk of Parkinson disease using three polymorphisms in two genes, These findings suggest that increased iron levels in the blood are associated with a 3% reduction in the risk of Parkinson disease for every 10 µg/dl increase in iron. This finding is important as it suggests that increased blood iron levels may have a protective effect against Parkinson disease, although the underlying mechanism remains unclear. Furthermore, although mendelian randomization is an increasingly used approach to address the issue of classical confounding, there may be remaining confounding factors specific of mendelian randomization that may influence the interpretation of this study. Nevertheless, the results of this analysis have potentially important implications for future research into the prevention of Parkinson disease. Further studies on the underlying mechanisms are needed before any specific treatment recommendations can be proposed.http://dx.doi.org/10.1371/journal.pmed.1001462.Please access these Web sites via the online version of this summary at Neurological Disorder and Stroke, MedlinePlus, and NHS Choices have several pages with comprehensive information on Parkinson diseaseThe National Institutes of explanation of mendelian randomization Wikipedia gives an  Iron is involved in fundamental biochemical activities, such as oxygen delivery, mitochondrial respiration, and DNA synthesis in almost all cell types. In the brain, iron is a cofactor for a large number of enzymes, including key enzymes of neurotransmitter biosynthesis, such as the tyrosine hydroxylase, which represents the rate-limiting enzyme of dopamine synthesis p = 0.09) 2: 93%; p<0.0001) makes it difficult to interpret these findings.Limited epidemiological evidence on the relationship between peripheral blood levels of iron and PD risk is available. A recent meta-analysis of ten studies, with a total of 520 PD cases and 711 controls, showed a trend for lower serum iron levels in PD patients compared with controls, although the difference in iron levels was not statistically significant  is an approach based on the use of genes as instrumental variables, which has been proposed to assess causality and provide estimates of the effect of modifiable intermediate phenotypes on disease unaffected by classical confounding or reverse causation, whenever randomized clinical trials are not feasible The MR estimate of the effect of the intermediate phenotype on the disease is derived from the estimates of the associations of the polymorphism with both intermediate phenotype and disease. MR, as any other instrumental variable approach, has low statistical power and therefore requires very large sample sizes The validity of the MR approach relies on the crucial assumption that the polymorphism acts on the disease only through the intermediate phenotype of interest and not through others (assumption of no pleiotropy) In this study, we provide evidence on the presence, direction, and magnitude of a causal effect of serum iron levels on PD risk by performing a MR study, based on iron data in 21,567 individuals from the general population and PD data from 20,809 PD cases and 88,892 controls. We used three polymorphisms as instruments in order both to increase statistical power by combining their MR estimates and to investigate the possible presence of pleiotropy.HFE, ENSG00000010704) gene and the transmembrane protease 6  gene have the strongest effects on serum iron in the general population of European ancestry HFE gene, rs1800562 (C282Y) and rs1799945 (H63D), which are not in linkage disequilibrium (HapMap CEU r2<0.01) and therefore represent independent signals of association, and one for the TMPRSS6 gene, rs855791 (V736A) (The selection of the genes modifying iron levels to be used as instruments in our MR study was based on published results showing that polymorphisms in the hemochromatosis ( (V736A) .Our MR approach was based on the use of aggregate results for both the gene–iron and gene–PD associations: for each polymorphism, we performed a meta-analysis of studies investigating its effect on iron levels and a meta-analysis of studies investigating its effect on PD risk, with no studies contributing to both meta-analyses (see next sections). Three separate MR estimates of the effect of iron on PD were obtained for the three polymorphisms, and they were subsequently pooled by meta-analysis to provide a single MR estimate. Heterogeneity between the three MR estimates was investigated to detect the possible presence of pleiotropy.HFE and TMPRSS6 on total serum iron levels was based on the findings of a recent GWA meta-analysis on iron parameters performed by the Genetics of Iron Status (GIS) Consortium , a database providing a regularly updated synopsis of genetic association studies performed in PD HFE, rs1800562 and rs1799945. A total of nine studies were included in our analysis for both rs1800562 Candidate gene studies were identified using PDGene  above the mean iron level associated with each copy of the allele.Study results for the candidate gene studies investigating the gene–PD risk association were obtained either from the PDGene website or directly from the original papers 2 statistics, a measure defined as the percentage of total variation in study estimates explained by heterogeneity rather than sampling error PD/iron is the (log) increase of PD risk by SD unit increase in iron (MR estimate), log ORPD/allele is the (log) increase in PD risk per allele (gene–PD association), and betairon/allele is the number of SDs above the mean iron level per allele (gene–iron association). The standard error of the MR estimate was derived using the Delta method PD/iron.As for the instrumental variable analysis, an MR estimate of the effect of iron on PD risk was obtained for each of the three instruments separately, and the three estimates were combined using an inverse-variance-weighted fixed-effect meta-analysis. We evaluated the presence and magnitude of heterogeneity across the three instruments with the I2 is the variance of iron blood levels explained by the genetic variant and n is the sample size for the gene–iron association. We also evaluated the overall F statistics for the three combined instruments assuming that their effects were independent, as are expected to be given that the three gene variants are not in linkage disequilibrium.We evaluated the strength of each instrument using the F statistics, which is a function of the magnitude and precision of the genetic effect on the biomarker (iron):A sensitivity analysis was performed to investigate the possible impact on our findings of population stratification in any of the studies included in the gene–iron or gene–PD analyses, by excluding studies which had not adjusted for population stratification.All analyses were performed using Stata 10 (StataCorp LP).p = 4.0×10−77) for each copy of the A allele of HFE rs1800562, 0.19  for the G allele of HFE rs1799945, and 0.19  for the G allele of TMPRSS6 rs855791  per copy of the G allele. As shown in the Forest plot of the meta-analysis for this polymorphism . In particular, although the 23andMe study was based on self-reported disease status and therefore differed from the rest, its results were consistent with those of the other PD studies. The association with PD risk for the two polymorphisms in HFE was not statistically significant, with an OR of 0.97  for the A allele of rs1800562 and 0.99  for the G allele of rs1799945 , as suggested by their wide confidence intervals.All datasets available for the analysis of the effects of the three genetic polymorphisms on PD risk  were chemorphism , there ws1799945 . This mip = 0.001), representing the OR for PD per SD unit increase in iron , suggesting that the assumption of no pleiotropy might hold.The meta-analysis of the three MR estimates resulted in a statistically significant combined estimate of 0.88   .p = 0.001) relative reduction in PD risk per 10 µg/dl increase in iron. If we hypothesise increasing serum iron levels of one SD unit (38 µg/dl in our study) in a population of Caucasians older than 60, where PD risk is around 1% Our study shows a protective effect of serum iron levels on PD, with a 3%  and rs1799945 (H63D), are non-synonymous polymorphisms in HFE, a gene with well known effects in the modulation of iron blood levels V736A), is located in TMPRSS6, a gene whose role in iron regulation was demonstrated more recently HFE gene are responsible for most cases of hereditary hemochromatosis C282Y/C282Y) or compound heterozygous (C282Y/H63D) state. The C282Y variant prevents the altered HFE protein from reaching the cell surface and interacting with the transferrin receptor (TfR) H63D variant is as yet unclear, but some evidence suggests that it may alter an intramolecular salt bridge, possibly affecting the interaction of the HFE protein with the TfR TMPRSS6 V736A variant was found associated with iron-deficiency anemia TMPRSS6 rs855791 was by far the most influential and was the one driving the result of the meta-analysis of MR estimates from the three instruments. The wide confidence intervals of the MR estimates for HFE rs1800562 and rs1799945 suggest that the power of their MR analysis was very limited due to their low allele frequency. This illustrates the importance of balancing the strength of the effect on the intermediate phenotype with allele frequency and statistical power when choosing the instruments for a MR study.The crucial aspect of a MR study, and more generally of any study based on an instrumental variable approach, is the choice of the gene (instrument) that needs to have a strong effect on the intermediate phenotype of interest. We used three polymorphisms as instrumental variables, since the use of multiple instruments influencing the intermediate phenotype of interest can increase the statistical power of the MR analysis HFE or TMPRSS6 genotypes could influence PD risk through another mechanism that is independent of their effect on serum iron levels. Although we cannot completely exclude pleiotropic effects of the three polymorphisms used in our study because of incomplete knowledge of the underlying biology, we can indirectly investigate the presence of such effects through the simultaneous use of the three polymorphisms as multiple instruments. In a MR study, if all instruments are valid, their MR estimates should differ only as a result of sampling error A potential source of bias specific to MR studies is pleiotropy, whereby the Despite all the possible limitations discussed above, MR offers a valuable approach to derive causal effect estimates whenever randomized trials are very difficult to perform, as in the case of iron and PD. A trial investigating the long-term effect of changes in a subject's iron status, obtained by some means, on the risk of developing PD would require not only a very long follow-up but also a huge sample size, given the low frequency of the disease and the magnitude of the effect that might realistically be expected.In our study, the MR analysis to combine the OR of the gene–PD association with the effect of the gene–iron association was based on a Wald-type estimator, which works under a “rare disease assumption” that is appropriate in the case of PD. However, the use of a Wald-type estimator for the MR analysis of binary outcomes represents only an approximate method and may produce biased MR estimates In summary, our MR study suggests a causal association between increased serum iron levels and decreased risk of developing PD, suggesting that disrupted iron metabolism may be an important factor in the pathogenesis of PD. However, further research is needed to elucidate the pathophysiological mechanism of action underlying our findings. The effect of dietary iron or drugs capable of altering the balance between serum iron and iron storage compartments, might prove to be suitable to test in experimental models. The development of such disease models is therefore necessary before any public health or clinical recommendation can be made for primary prevention in subjects at high risk of developing PD.Figure S1Forest plot of the meta-analysis of the studies included for the effect of HFE rs1800562 on PD risk. The boxes indicate the genetic (additive) effects of individual studies, with the size of the box being inversely proportional to the variance and horizontal lines indicating 95% confidence intervals. The diamond indicates the pooled effect estimate, obtained using inverse-variance weighted fixed-effect meta-analysis, and its 95% confidence interval. The full vertical line shows the value for no effect, as opposed to the dashed line indicating the estimated pooled effect.(TIF)Click here for additional data file.Figure S2Forest plot of the meta-analysis of the studies included for the effect of HFE rs1799945 on PD risk. The boxes indicate the genetic (additive) effects of individual studies, with the size of the box being inversely proportional to the variance and horizontal lines indicating 95% confidence intervals. The diamond indicates the pooled effect estimate, obtained using inverse-variance weighted fixed-effect meta-analysis, and its 95% confidence interval. The full vertical line shows the value for no effect, as opposed to the dashed line indicating the estimated pooled effect.(TIF)Click here for additional data file.Figure S3Forest plot of the meta-analysis of the studies included for the effect of TMPRSS6 rs855791 on PD risk. The boxes indicate the genetic (additive) effects of individual studies, with the size of the box being inversely proportional to the variance and horizontal lines indicating 95% confidence intervals. The diamond indicates the pooled effect estimate, obtained using inverse-variance weighted fixed-effect meta-analysis, and its 95% confidence interval. The full vertical line shows the value for no effect, as opposed to the dashed line indicating the estimated pooled effect.(TIF)Click here for additional data file.Figure S4Sensitivity analysis: Forest plot of the mendelian randomization estimates after exclusion of nine studies from the PDGene dataset that had not adjusted for population stratification see .(TIF)Click here for additional data file.Table S1Characteristics and sample size of the individual studies included for the gene–iron association. In all studies, the analyses were adjusted for age and sex, as well as for the first five MDS  or principal components to control for population stratification.(DOC)Click here for additional data file.Table S2Characteristics and sample size of the individual studies included for the gene–PD association.(DOC)Click here for additional data file.Table S3Gene–iron association: GIS-consortium meta-analysis. The effect size for the genetic effects on iron levels is expressed as number of SDs from the mean (Z-scores).(DOC)Click here for additional data file.Table S4Gene–PD association: meta-analysis of all available candidate gene and GWA studies.(DOC)Click here for additional data file.Text S1Detailed description of the studies included in the three GWA investigations of PD risk.(DOC)Click here for additional data file.


APOA5) gene −1131T>C (rs662799) has been suggested to be involved in the pathway of lipid homeostasis and the development of metabolic syndrome (MetS). However, the findings are not consistent. To systematically evaluate the associations between −1131T>C polymorphism and fasting lipid parameters and the risk of MetS, we conducted a case-control study in a Chinese population and a meta-analysis. The findings from 1840 Chinese participants indicated that the C allele carriers had significantly higher fasting total cholesterol (TC), triglycerides (TG) and lower HDL-cholesterol (HDL-C) than the TT homozygotes carriers. The −1131C allele was also found to be significantly associated with increased risk of MetS   =  1.15, 1.69) compared to the TT homozygotes. In the meta-analysis of 51,868 participants from 46 East Asian studies, 26 European studies and 19 studies of other ethnic groups, the −1131C allele was associated with higher fasting TC (weighted mean difference (WMD)  =  0.08 mmol/L, 95% CI  =  0.05, 0.10, P = 1.74×10−9), TG , LDL-cholesterol (LDL-C) , and lower HDL-C , respectively. Based on 12 studies with 5,573 MetS cases and 8,290 controls from 5 East Asian studies, 5 European studies and 2 studies of other ethnic groups, the −1131C allele was associated with increased risk of MetS with an OR (95% CI)  =  1.33  in the overall population, 1.43  in East Asian and 1.30  in European populations. In conclusion, the −1131C allele may be associated with elevated levels of fasting TG, TC, LDL-C and decreased HDL-C, and increased risk of MetS, especially in East Asians.The apolipoprotein A5 ( APOA5) gene has been found to be associated with the increased risk of MetS. The +1891T>C (rs2266788) in APOA5 gene was reported associated with MetS in a European GWAS APOA1/C3/A4/A5 gene cluster region, rs964184, was identified associated with MetS in Finnish populations by a GWAS APOA5 is considered a potential biomarker for MetS.Metabolic syndrome (MetS), characterized by visceral obesity, dyslipidemia, hypertension and hyperglycemia, has become one of the major public health challenges worldwide APOA5 gene is part of the APOA1/A4/C3/A5 gene cluster on 11q23, identified by comparative sequencing analysis APOA5 gene consists of four exons and three introns. It codes a protein with 369 amino acids. The human APOA5 gene is exclusively expressed in liver and its product apoA-V can be detected in very low-density lipoprotein (VLDL), high-density lipoprotein (HDL) and chylomicrons. It plays an important role in regulating plasma triglyceride levels in both human beings and mice −1131T>C, in the promoter region of APOA5 gene, is the most extensively studied variant. The −1131T>C has been reported to be associated with hypertriglyceridemia −1131T>C polymorphism with triglycerides (TG) have been reported in African-American or European women by Pennachio and Rubin −1131C allele has also been reported to be associated with higher low-density lipoprotein-cholesterol (LDL-C) APOA5 gene −1131T>C polymorphism and fasting lipid parameters and the risk of metabolic syndrome, we conducted a case-control study in a Chinese population and a meta-analysis based on currently reported studies.The case-control study was conducted from 2010–2011. Our study population was unrelated individual residents from Xiaoshan area, Zhejiang, P.R. China. A total of 905 MetS cases and 935 controls were recruited based on the following criteria. MetS was diagnosed according to the criteria of International Diabetes Federation (IDF) All participants were given and signed the written informed consent form and the study protocol was approved by the Institutional Review Board of School of Public Health, Zhejiang University.2). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) was measured while subjects were in the sitting position after 15 minutes of rest. The concentrations of fasting total cholesterol (TC), TG, LDL-C, and HDL-C in the plasma were analyzed by enzymatic methods with commercially available kits on a Hitachi 7180 Autoanalyzer . Glucose was analyzed by a glucose oxidase method with the Beckman Glucose Analyzer .Height, weight and WC were measured while the subjects were dressed only in their undergarment and did not wear shoes after an overnight fast. At the same time, 2 ml of peripheral venous blood was drawn from each subject. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters (kg/m+1891T>C (rs2266788) was included in this database. The −1131T>C was found to be in strong linkage disequilibrium with the +1891T>C (rs2266788) in East Asians (CHB plus JPT) from HapMap phase II database . Therefore, the genotype information of the −1131T>C was imputed using MACH 1.0 2-hat)  =  0.8488 was considered well imputed.An ongoing GWAS project on MetS has been performed for all the subjects with Illumina Beadchip OmniExpress . The genotype of the x2 test with PLINK software (version 1.0.6) APOA5 genotypes with age, gender as covariates.Risk of MetS was evaluated under the unconditional logistic regression model and the genotype under assumptions of additive and dominant genetic models, respectively. Statistical power was calculated by Quanto version 1.2.4 (http://hydra.usc.edu/gxe/). For disease trait, power was calculated on the basis of cases sample, a ratio of controls vs. cases, allelic frequency, genetic effect under an inheritance model, a significance level, and the disease prevalence in population level. For quantitative trait, power was calculated on the basis of sample size, allelic frequency, population mean, standard deviation, and R squared (RG). A P value ≤0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS 16.0 .Continuous variables were expressed as mean ± standard deviation (SD). Differences between case and control groups were estimated by two independent variable t-test for continuous variables and by Chi-square test for categorical variables. The departure from Hardy–Weinberg equilibrium was computed by the goodness-of-fit APOA5”, or “apolipoprotein A5”, in combination with “polymorphism”, or “variant”, or “SNP”, or “mutation” without language restriction. The last search was updated in March 2012. These searches were supplemented by scanning reference lists and correspondence with authors.We conducted a comprehensive search of Pubmed, HugeNavigator, Medline and the Chinese National Knowledge Infrastructure Database (CNKI), using the following keywords: “−1131T>C variant of APOA5 gene was studied; (3) the indices of outcomes were fasting TC or TG or LDL-C or HDL-C, or MetS; (4) if there were multiple studies from the same population, only the paper with the largest population would be extracted. Animal studies, reviews, case reports and simply commentaries were excluded.The inclusion criteria for studies were as follows: (1) the participants were greater than 18 years old; (2) the Two investigators (Chunxiao Xu and Rongpan Bai) extracted data independently. The discrepancies were resolved by discussion with a third author (Dandan Zhang). The following data were collected from each study: first author's name, year of publication, ethnicity, study population, sex, health condition, sample size, genotype and allele distributions among participants with and without MetS, and mean with standard deviation (SD) or standard error by genotypes.−1131T>C and MetS or lipid parameters were analyzed with a dominant model. For lipid parameters, weighted mean difference (WMD) and its 95% confidence interval (CI) were calculated. For dichotomous data on disease status, pooled odds ratio (OR) and 95% CI were calculated in overall and subgroup populations. The heterogeneity among the studies was assessed by the Chi square-test based Q-statistic and 2I test. A significant level, α, was set as 0.1 because Q-statistic is considered underpowered. P value greater than 0.1 for the Q-statistic implies a lack of heterogeneity among studies. On this condition, the fixed-effect model (the Mantel–Haenszel method) APOA5 −1131T>C polymorphism and lipid levels, random-effect model was employed. Hardy-Weinberg equilibrium (HWE) in each group was estimated by the Chi-square test. Studies deviating from HWE were not excluded because there was no benefit in excluding these studies from genetic meta-analysis The associations between the P value less than 0.05.We also conducted subgroup analysis by excluding studies deviating from HWE. Galbraith plot was used to detect potential sources of heterogeneity. As for publication bias, Eegg's linear regression and Begg's funnel plots were performed. And Duval and Tweedie's trim and fill method P < 0.001). No statistical difference in gender was found between cases and controls (P > 0.05). BMI, WC, SBP, DBP, TC, TG, LDL-C, and fasting plasma glucose in case group were significantly higher, whereas HDL-C was significantly lower than those in control group .The basic characteristics of the subjects between 905 MetS cases and 935 controls are summarized in −1131T>C between case and control groups are shown in P = 0.139). The frequency of the −1131C allele in MetS cases was significantly higher than that in controls . Under the dominant model and adjusted for age and gender, the carriers with the −1131C allele were found to have an increased risk for MetS . Additionally, significantly increased risk of MetS was also found under the additive model, with per-C allele OR of 1.32 (1.08–1.62).Genotype distributions and allele frequencies of the – 1131T>C genotype. Among the combined overall population, the C allele was significantly associated with increased plasma concentrations of TG (P < 0.001) and TC (P = 0.024), and decreased level of HDL-C (P = 0.007). In case group, the −1131C allele was also related to higher TC, TG, LDL-C and lower HDL-C while significance was only observed for TG (P = 0.001). In control group, although the associations between the −1131T>C and lipids were not statistically significant, the trends were consistent.APOA5 gene-lipid associations APOA5 gene-MetS association Fifty-eight eligible papers were extracted and our own case-control study was included for meta-analysis . Ninty-o−1131C carriers had an increased TC by 0.08 mmol/L  compared with the TT group , man, and woman subgroups.The TT group . Subgrou−1131C carriers increased 0.30 mmol/L  compared with the TT group  compared with the TT group (Mean plasma LDL-C levels for TT group . Weak buTT group .P = 1.88×10−21) compared with the TT group : pooled OR =  1.33, 95% CI  =  1.16, 1.53, P =  0.00004 : 1.30 . When stratifying by study design, significant association was detected in both population-based  and hospital-based studies   .−1131C allele and HDL-C in CHD patients . However, an asymmetry funnel plot detected for the −1131T>C and serum TG levels suggested the presence of publication bias. After adjustment with trim and fill method, the pooled WMD was reduced to 0.18  compared with unadjusted pooled WMD, but this was still statistically significant indicating that the results were statistically robust.With Begg's funnel plot and Eegg's test, no publication bias was detected among the studies on the association of the −1131T>C of APOA5 gene and lipid levels and the risk for MetS in a case-control study in a Chinese population and meta-analysis. In the case-control study, the individuals with the −1131C allele had a higher level of fasting TG in MetS cases, higher TC, TG and lower HDL-C and 40% increased MetS risk in the overall Chinese population. The meta-analysis of gene-lipid association in 51,868 participants showed that the −1131C allele was associated with higher levels of fasting TG, TC, LDL-C and lower level of HDL-C. Moreover, the gene-MetS association meta-analysis pooling data from 11 previous studies and the current containing 13,863  subjects also confirmed that individuals with the −1131C allele had a 33% increased risk to develop MetS.In this study, we evaluated associations between the genetic variation of the APOA5 gene are among the strongest known genetic determinants of plasma TG concentration, and also have been reported to be associated with higher TC, LDL-C and lower HDL-C concentration APOA5 gene can help to clarify the relevance of the APOA5 pathway to lipid metabolism and MetS risk. The −1131T>C located in the promoter region of APOA5 gene, might down-regulate APOA5 mRNA translation, which in turn results in lower apo A5 levels, and then leads to higher levels of TG −1131T>C in the reduction of gene expression remains to be studied. The −1131T>C has been observed in modest to strong linkage disequilibrium with another locus, +1891T>C, in different populations +1891T>C was associated with MetS in a European GWAS conducted by STAMPEED Consortium −1131T>C and +1891T>C were not in the same haplotype block in their study population. Till now, few variants in or near this gene have been reported associated with MetS by relative few European GWASs. Another intergenic locus in APOA1/C3/A4/A5 gene cluster region, rs964184, was identified associated with MetS in Finnish populations by a GWAS Polymorphisms in or near the −1131T>C was imputed according to directly genotyped +1891T>C in our GWAS data. With a strong disequilibrium, the observed genotypes provide adequate information about the unobserved genotypes, and the imputation algorithm can infer genotypes with high confidence −1131T>C was found to be in strong linkage disequilibrium with the +1891T>C in East Asians from HapMap database. And the genotyping quality score (R2-hat)  =  0.8488 suggested that the −1131T>C was well imputed.The genotype of the 1131T>C was related to TC, TG and HDL-C, however, unrelated to LDL-C. These findings are similar to a previous meta-analysis mainly consisting of Europeans conducted by Triglyceride Coronary Disease Genetics Consortium and Emerging Risk Factors Collaboration, in which they found that for every C allele of the APOA5 –1131T>C genotype inherited, mean TG level was higher and mean HDL-C level was lower et al.−1131T>C on TG and also found a positive association.Meta-analysis in 11,583 European individuals showed that the −−1131C and LDL-C in East Asians. A total of 24,563 East Asian individuals were included in the meta-analysis which ensured adequate power. Mean plasma LDL-C level for the −1131C carriers was 0.04 mmol/L  higher than that for the TT carriers (P = 0.001). There are some possibilities for the discrepancy between Europeans and East Asians. First, genotypic effects could be ethnicity specific. Compared with Europeans , the minor allele frequency of the −1131T>C among East Asians was much higher . Second, there are different lifestyle environmental factors such as diet especially animal food consumption and alcohol use which have been reported to play a role in modifying the effect of APOA5 on plasma lipids To our knowledge, this is the first study to conclude a positive association between the et al.−1131C allele was associated with increased MetS risk in overall population and in Europeans. Our larger sample with 5,573 cases and 8,290 controls with 5 studies in East Asians and 5 studies in Europeans and 2 in other ethnic studies provided higher OR: 1.33  and the novel finding that positive association was only detected in East Asians but not in Europeans. To our knowledge, it was the first meta-analysis to provide significant evidence for this association in East Asian populations. We noted that a study in Europeans by Dallongeville ser19trp, C56G) was reported to have a positive association with MetS was included in the Povel's. This study may be the source of the inconsistent conclusions in Europeans between our meta-analysis and the Povel's. In our study, inconsistency between Europeans and East Asians can be explained by diverse gene-environment interactions in different ethnic groups MetS in our case-control study was defined by IDF while most of our available studies were defined by NCEP ATP III −1131C allele carriers and non-carriers. With subgroup analysis, heterogeneity was removed in some subgroups but still existed in others. By using Galbraith plot, outlier studies contributing to heterogeneity were identified. After exclusion of these outlier studies, heterogeneity was effectively disappeared or decreased while the recalculated results remained similar with the exception of the association between the −1131C allele and HDL-C in CHD patients. This indicated that more studies should be conducted to further examine the association of this polymorphism with HDL-C in CHD patients.Heterogeneity was detected among studies comparing the levels of TG and HDL-C and risk of MetS between the −1131T>C polymorphism and plasma TG concentration. It is known that the existence of publication bias may change the conclusion of a meta-analysis. Here using Duval and Tweedie's trim and fill method, a pooled WMD adjusted for publication bias was calculated. And the result with slightly lower mean TG level remained significant suggesting the reliability of the whole pooled result.Publication bias was only observed in studies on the association between the APOA5 −1131C allele was associated with elevated levels of fasting TG, TC, LDL-C and decreased HDL-C, and associated with increased risk of MetS, especially in East Asians. Second, it was the first meta-analysis to provide significant evidence for the associations of the APOA5 −1131T>C with LDL-C and MetS in East Asian populations. Third, our study adds additional evidence of the effect size of the −1131T>C on lipid levels in East Asians which has not been investigated either by Triglyceride Coronary Disease Genetics Consortium and Emerging Risk Factors Collaboration or by Saleheen. Fourth, the statistical power of both our case-control study and meta-analysis is adequate  which supports the reliability of the results.There are several major strengths in our study. First, our study confirmed and extended the previous findings that the APOA5 −1131T>C variant on plasma lipid levels and the prevalence of metabolic syndrome, there are still many other APOA5 SNPs, gene-gene and/or gene-environment interactive factors needed to be considered. For example, polymorphisms in the APOC3 gene have been shown to interact with APOA5 gene and affect plasma TG concentration APOA5 gene which may be related to our studied outcomes. Third, the genotype of the −1131T>C was imputed instead of directly genotyped. Therefore we make a cautious conclusion.Meanwhile, there are potential limitations in this study. First, although we only explored the effect of APOA5 −1131C allele may be associated with increased levels of fasting TG, TC, LDL-C and decreased HDL-C, and associated with increased risk of MetS, especially in East Asians.In conclusion, the Figure S1Flow chart of study identification.(TIF)Click here for additional data file.Figure S2−1131T>C and TC, TG, LDL-C and HDL-C under dominant model (CC/CT vs. TT). Each point represents a separate study for the indicated association. SE (WMD), standard error (weighted mean difference). (A) The funnel plot comparing the differences in TC; (B) The funnel plot comparing the differences in TG; (C) The funnel plot comparing the differences in LDL-C; (D) The funnel plot comparing the differences in HDL-C.Funnel plot for (TIF)Click here for additional data file.Figure S3−1131T>C and metabolic syndrome under dominant model (CC/CT vs. TT). Each point represents a separate study for the indicated association. SE (logor), standard error of log (odds ratio).Funnel plot for (TIF)Click here for additional data file.Figure S4APOA5 −1131T>C polymorphism and plasma TG under dominant model (CC/CT vs. TT).Galbraith plot of (TIF)Click here for additional data file.Figure S5APOA5 −1131T>C polymorphism and plasma HDL-C under dominant model (CC/CT vs. TT).Galbraith plot of (TIF)Click here for additional data file.Figure S6APOA5 −1131T>C polymorphism and risk of metabolic syndrome under dominant model (CC/CT vs. TT).Galbraith plot of (TIF)Click here for additional data file.Table S1Characteristics of the individual included studies with fasting lipid levels as outcome.(DOC)Click here for additional data file.Table S2−1131T>C.Plasma lipid levels by genotypes of individual studies included in the meta-analysis for the (DOC)Click here for additional data file.Table S3APOA5 −1131T>C with metabolic syndrome.Studies included in the meta-analysis of the association of (DOC)Click here for additional data file.Table S4APOA5 −1131T>C on fasting plasma lipids and metabolic syndrome risk.Subgroup analysis in a meta-analysis of the effects of (DOC)Click here for additional data file.Table S5APOA5 −1131T>C on fasting plasma lipids and metabolic syndrome risk (excluding the outlier studies).Meta-analysis of the effects of (DOC)Click here for additional data file.


In addition, microRNAs affect all facets of the immune/inflammation responses to infection, which may control HPV clearance. We thus hypothesized that microRNA polymorphisms modify the association between HPV16 seropositivity and OSCC risk.Both microRNAs were genotyped and HPV16 serology was determined in 325 cases and 335 matched controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using univariate and multivariable logistic regression models.Four single-nucleotide polymorphisms in miR146 rs2910164 GG genotype and HPV16 seronegativity, risk of OSCC was increased among those with CG or CC genotype and HPV16 seronegativity , GG genotype and HPV16 seropositivity , and CG or CC genotype and HPV16 seropositivity . Similar results were found for miR149 rs2292832, miR196 rs11614913, and miR499 rs3746444. Analyses stratified by tumor sites and smoking status showed that each polymorphism significantly increased the risk of HPV16-associated squamous cell carcinoma of the oropharynx (SCCOP), and such effect modification was particularly prominent in never smokers.Overall, each polymorphism had no significant main effect on OSCC risk. Compared with the risk among individuals with both microRNA polymorphisms modify the risk of OSCC associated with HPV16 seropositivity, particularly in patients with SCCOP and never smokers. Larger studies are needed to verify our findings.Our results indicate that  Oral squamous cell carcinoma  accounts for the majority of head and neck malignant tumors and is one of the most common malignancies worldwide. It has been estimated that approximately 36,540 new cases of OSCC will be diagnosed and 7,880 individuals will die from these cancers in 2012 in the United States MicroRNAs are highly conserved, single-stranded, short, noncoding RNAs of about 22 nucleotides that regulate gene expression through completely or partially base pairing with target mRNAs at the 3′-untranslated region, resulting in mRNA cleavage or translational suppression microRNAs play key roles in a broad range of physiologic and pathologic processes microRNAs remain largely unclear, previous studies have indicated that microRNAs may participate in tumorigenesis, functioning as tumor suppressors and/or oncogenes microRNAs may be involved in regulation of the immune and inflammatory response systems microRNAs may affect susceptibility to HPV-associated OSCC.hsa-146a was over-expressed in SCCHN cell lines and tumor tissues and hsa-mir-149 was down-regulated in squamous cell carcinoma of the tongue and hsa-mir-146a polymorphism may also affect the miRNA expression miR146 G>C, miR196 C>T, and miR499 T>C may affect cancer risk and prognosis miR146 rs2910164, miR149 rs2292832, miR196 rs11614913, and miR499 rs3746444) and risk of head and neck cancers, but no significant association between each SNP and cancer risk was found microRNAs in regulating the immune and inflammatory response systems and thereby mediating HPV infection, we hypothesized that genetic variants in these four microRNAs alter the association between HPV16 seropositivity and risk of OSCC. To test this hypothesis, we evaluated the joint effect of HPV16 serology and these four common microRNA SNPs on risk of OSCC.Previous studies have demonstrated that Patients with incident OSCC were recruited as part of an ongoing molecular epidemiology study of head and neck cancers through the Head and Neck Center at The University of Texas MD Anderson Cancer Center between December 1996 and October 2002. All patients were recruited without restrictions on age, sex, cancer stage, or histology and had newly diagnosed, histopathologically confirmed, untreated OSCC. Excluded from participation were patients with second primary tumors; primary tumors of the sinonasal tract, nasopharynx, hypopharynx, or larynx; primary tumors outside the upper aerodigestive tract; cervical metastases of unknown origin; and histopathologic diagnoses other than SCC. In addition, patients who had received a blood transfusion in the past 6 months or who were receiving immunosuppressive therapy were excluded. Among patients initially contacted for participation, approximately 95% of eligible incident cases agreed to participate in the study.A pool of cancer-free subjects was recruited from the Kelsey-Seybold Foundation, a multispecialty physician practice with multiple clinics throughout the Houston metropolitan area, and from healthy visitors who accompanied cancer patients to outpatient clinics at MD Anderson Cancer Center but were genetically unrelated to the cases. In this pool of cancer-free controls, each individual was first surveyed by means of a short questionnaire to determine his or her willingness to participate in research studies. Each eligible subject was subsequently interviewed to collect demographic and epidemiologic information, such as age, sex, ethnicity, smoking status, and alcohol consumption status. The overall response rate for the controls was approximately 78%. Excluded from participation were individuals with history of any cancer, history of immunosuppressive therapy, or blood transfusion in the past 6 months.Smoking status was categorized as “ever smokers” (those who had smoked more than 100 cigarettes in their lifetime) and “never smokers” . Drinking status was categorized as “ever drinkers”  and “never drinkers” (those who never had such a pattern of drinking). Both Institutional Review Boards of UT MD Anderson Cancer Center and Kelsey-seybold approved the study, and written informed consent was obtained from all study subjects. In this study, 335 cancer-free controls were selected from the pool of potential controls by frequency matching with the patients by age (±5 years), sex, and smoking and drinking status. To avoid confounding factors due to ethnic characteristics, all study subjects included were non-Hispanic whites, who accounted for the vast majority of the cases.HPV16 L1 virus-like particles generated from recombinant baculovirus-infected insect cells were used to test for antibody against HPV16 L1 capsid protein in the plasma of study participants by using a standard enzyme-linked immunosorbent assay, as described previously miR146 rs2910164, miR149 rs2292832, miR196 rs11614913, and miR499 rs3746444. The microRNA SNPs, primers, and restriction enzymes  used for each SNP are shown in From each blood sample, a leukocyte pellet was obtained from the buffy coat by centrifugation of 3 mL of whole blood. The pellet was used for genomic DNA extraction with the DNA Blood Mini Kit  according to the manufacturer's instructions. A polymerase chain reaction-restriction fragment-length polymorphism assay was used to amplify the fragments that contained SNPs of microRNA genotypes, and HPV16 serology between cases and controls were evaluated using the chi-square test. To evaluate the association of HPV16 serologic status and microRNA genotypes with OSCC risk, odds ratios (ORs) and 95% confidence interval (CIs) were calculated using both univariate and multivariate logistic regression analyses. Logistic regression was also used to assess the potential interaction effects by evaluating departures from the models of additive and multiplicative interactions between selected variables. A more-than-additive interaction was suggested when OR11>OR10+OR01−1, for which OR11 = OR when both factors were present, OR10 = OR when only factor 1 was present and OR01 = OR when only factor 2 was present. A more-than-multiplicative interaction was suggested when OR11>OR10×OR01. We assessed the interaction by reporting the P values from the Wald test for testing the coefficients  were different from 0, where the interaction term consisted of the product of the two variables: microRNA polymorphism and HPV16 seropositivity. We also assessed the joint effects of HPV16 serology and microRNA genotypes on OSCC risk, and the joint effects were further stratified by tumor site and smoking status. Statistical Analysis System software  was used for all statistical analyses. All tests were two-sided, and P<0.05 was considered the cutoff for statistical significance.The distributions of selected demographic variables, tobacco smoking, alcohol consumption, P>0.05). However, we observed that HPV16 seropositivity was significantly more common in patients than in controls (P<0.001). HPV16 seropositivity was associated with risk for OSCC , particularly for SCCOP , but not for oral cavity cancers , after adjusting for age, sex, smoking status, and drinking status.The final analysis included 325 OSCC cases and 335 controls. Of the 325 cases, 188 (57.8%) had SCCOP, and 137 (42.2%) had SCC of the oral cavity. The distributions of demographic characteristics and known OSCC risk factors are summarized in microRNA SNPs among the controls were all in agreement with the Hardy-Weinberg equilibrium . Overall, we did not find any statistically significant association of any of the four microRNA SNPs with the risk of OSCC, but we did find that miR146 rs2910164 and miR499 rs3746444 had a minor effect on the risk of SCCOP , and individuals with the miR499 rs3746444 combined CT and CC genotypes had a higher risk of SCCOP than individuals with the TT genotype .The genotype distributions of the four of SCCOP Table 3.microRNA genetic variants. Specifically, compared with individuals with both miR146 rs2910164 GG genotype and HPV16 seronegativity, those with both GG genotype and HPV16 seropositivity had an increased risk of OSCC , and the risk was even higher among those with both CG or CC genotype and HPV16 seropositivity . In contrast, compared with individuals with both miR149 rs2292832 CT or TT genotype and HPV16 seronegativity, those with both CT or TT genotype and HPV16 seropositivity had an increased risk of OSCC , and the risk was even higher among those with both CC genotype and HPV16 seropositivity . Similar results were observed for the associations between miR196 rs11614913 and miR499 rs3746444 SNPs and risk of HPV16-associated OSCC .As shown in ted OSCC Table 4.microRNA SNPs on the risk of HPV16-associated OSCC were confounded by smoking status, we performed stratified analyses of joint effects of HPV16 seropositivity and microRNA variants on risk of OSCC by smoking status (In order to explore whether the effects of g status Table 6.microRNAs on risk of OSCC, but we did observe a significant association between miR146 rs2910164 and miR499 rs3746444 and a moderately increased risk of SCCOP. However, we found that the joint effect of HPV16 seropositivity and each of these microRNA SNPs increased the risk of OSCC, although we did not observe any significant interaction for such joint effect on risk of OSCC. Moreover, such effect modification was more pronounced for SCCOP as opposed to SCC of oral cavity and in never smokers than in ever smokers. Our results are in agreement with the characteristics of SCCOP associated with HPV infection, indicating that microRNA SNPs might play a role in the development of HPV16-associated SCCOP.In this study, overall, we did not observe a significant main effect of each SNP of these MicroRNAs act as omnipresent regulators of gene expression and are involved in many cellular processes, including inflammation and immune responses. Recent studies have demonstrated that microRNAs participate in mediating inflammatory and cytokine signaling as well as innate and acquired immune response to viral infection by targeting critical elements in inflammatory signaling pathways MicroRNAs also modify viral-host interactions, which play central roles in the development and progression of infection-associated tumors.microRNAs may lead to individual variations in immune function, inflammation, and apoptosis that modify viral immune escape, antiviral defense, and evasion of apoptosis, leading to modification of the risk of infection-associated cancers. Numerous studies have shown that some microRNAs are up-regulated, while other microRNAs are down-regulated in head and neck cancers microRNAs may be associated with the development, progression, and prognosis of head and neck cancers Therefore, functional genetic polymorphisms of microRNA SNPs and the risk of head and neck cancers. One study indicated that the miR196 rs11614913 variant reduced the risk of head and neck cancers miR146 rs2910164, miR149 rs2292832, and miR196 rs11614913 did not modify the risk of head and neck cancers independently of HPV infection but that miR499 rs3746444 moderately reduced the risk of head and neck cancers microRNA SNPs and HPV16 infection on OSCC risk. HPV16 infection has recently been identified as one of the primary etiologic factors for causes of SCCOP, but oral cavity and laryngeal cancer mainly result from tobacco and alcohol consumption. In our current study, although we had a smaller study size, we found that microRNA SNPs and HPV16 seropositivity may function jointly in the development of OSCC, particularly in patients with SCCOP and in never smokers.Recently, several authors have reported associations between microRNA genetic variants and HPV16 infection jointly play a role in the development of OSCC has not been fully clarified, a joint effect of microRNAs and HPV16 infection on susceptibility to OSCC is biologically plausible. HPV16 is oncogenic: it encodes viral oncoproteins E6 and E7, which inhibit p53 and Rb cell cycle tumor suppressors. MicroRNA variants might functionally affect expression of genes involved in myriad cellular processes, including inflammation and immune response pathways, subsequently controlling the host's ability to clear HPV and HPV's ability to escape immune surveillance microRNA variants might act jointly in the development of OSCC. However, further studies are required to validate the hypothesized functionality of these SNPs.Although the precise mechanism by which the common microRNA SNPs and HPV seropositivity on the risk of OSCC was much stronger in never smokers than in ever smokers. These data further support the notion that risk genotypes of the four common SNPs of microRNAs may be involved in the development of HPV-associated OSCC in non-Hispanic white never smokers. However, the modifying effect of each of microRNA SNPs on risk of OSCC associated with HPV16 was not statistically significant. This lack of significance could be either because there was no such interaction effect in these subgroups or because the small sample sizes in each substratum limited the statistical power to detect a significant interaction effect. Therefore, the significance and degree of such interaction in each subgroup needs to be further investigated in future studies with larger sample sizes.In the present study, our analysis stratified by tumor site showed that the effect modification was more pronounced for SCCOP than for oral cavity SCC, reinforcing the concept that there are differences in the etiology of cancers of the oropharynx and oral cavity. Additionally, the analysis further stratified by smoking status for each SNP showed that the joint effect of Although the present study minimized potential confounding factors, several limitations should be considered. First, a possible selection bias in patient recruitment cannot be ruled out as our study was a hospital-based case-control study and as the cases and controls were not selected from the same target population. In addition, only non-Hispanic white subjects were included in our study. It is therefore uncertain whether these results are generalizable to other ethnic populations. Second, the effects of potential demographic confounding factors were minimized by adequate frequency matching on age, sex, smoking status, and alcohol use, but stratified analyses included a limited number of individuals in each subgroup. As such, the role of these SNPs and HPV16 in OSCC will require confirmation in larger studies in different populations. Third, because individual patients may have had differences in their immune response to HPV16 infection or antibody instability, HPV16 seropositivity may not reflect the tumor's true HPV16 status, leading to possible misclassifications of some patients as HPV16 seronegative who had tumors that actually were HPV16 DNA positive. This misclassification could result in a major selection bias for the estimates of association. Finally, lack of information on changes in sexual practices in current study did not allow us to evaluate its potential influence on risk of HPV-associated OSCC, particularly SCCOP. Therefore, our future studies should include the patients with tumor HPV status and sexual behaviors to further evaluate our findings once such information become available. Nevertheless, the use of serologic status may provide a possibility to include a cancer-free control group for the present case-control study design.microRNA variants may modify the risk of HPV16-associated OSCC, particularly in patients with SCCOP and in never smokers. However, further prospective studies with larger sample sizes are needed to validate our findings.In conclusion, our results indicate that


GNB3 C825T polymorphism and hypertension or stroke. The results of these studies were inconsistent; therefore, we performed a meta-analysis to clarify these discrepancies.Several epidemiological studies have evaluated the association between the We systematically searched the PubMed, Embase, Web of Science, CNKI, and CBM databases, and manually searched reference lists of relevant papers, meeting abstracts, and relevant journals. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for dominant, recessive, and allelic models. A fixed or random effects model was separately adopted depending on study heterogeneity. Subgroup and sensitivity analyses were performed to detect study heterogeneity and examine result stability, respectively. Publication bias was tested using funnel plots, the Egger's regression test, and Begg's test.We screened 66 studies regarding hypertension and eight concerning stroke. A combined analysis showed that only the allelic model found a marginal association with hypertension  and female gender . However, no comparison models found an association with stroke . Sensitivity analysis suggested that all models did not yield a relationship to hypertension or stroke among Asians. Besides, there was a lack of statistical association with hypertension in Caucasians, which maybe due to a small sample size. When we restricted the included studies to normal populations according to the Hardy–Weinberg equilibrium, no association was found.There was no evidence indicating that the 825T allele or TT genotype was associated with hypertension or stroke in Asians or hypertension in Caucasians. However, further studies regarding Africans and other ethnicities are needed to identify further correlations. Hypertension is a major risk factor of stroke, cardiovascular disease, and end-stage renal disease and affects about 1 billion adults worldwide, including 3.8 million in Taiwan and 160 million in China Guanine nucleotide-binding proteins (G proteins) are key determinants of specific and temporal characteristics of many signaling processes and are expressed in all cells of the human body to primarily transduce signals from the cell surface into a cellular response. G proteins consist of α, β, and γ subunits and different genes encode for 18 α subunits, 5 β subunits, and 12 γ subunits, which enable the formation of highly variable heterotrimers GNB3) gene encodes the Gβ3 subunit of heterotrimeric G proteins and is located on chromosome 12p13 and comprises 11 exons and 10 introns. A polymorphism  was found to be associated with a shortened splice variant of the Gβ3 protein that gives rise to enhanced signal transduction via pertussis toxin-sensitive G proteins The G protein beta polypeptide 3  criteria GNB3 C825T polymorphism and essential hypertension or stroke; (2) sufficient data on genotypic and allelic frequencies to determine an odds ratio (OR) with a 95% confidence interval (CI). If multiple publications reported the same or overlapping data, the most recent or complete study or the largest population was included in this meta-analysis as described by Little et al. Studies were screened that met the following criteria: (1) population-based or hospital-based case-control studies regarding the relationship between the 2 test. A p-value of <0.05 for the HWE was considered statistically significant.Data were independently extracted from each study by three investigators  following the above-mentioned inclusion criteria. Discordance was resolved by discussion or another reviewer (JCL) was consulted. The following data were collected from each of the selected studies: surname of the first author, year of publication, country of origin, population ethnicity, source of control, T allele frequency in controls, genotype variance in the cases and controls, and the Hardy–Weinberg equilibrium (HWE) using the χwww.ohri.ca/programs/clinical_epidemiology/oxford.asp). Scores were based on the selection, comparability, and exposure (case-control studies) or outcome (cohort studies) of the studies. To avoid selection bias, studies of poor quality were not rejected in this meta-analysis.The quality of each selected study was assessed independently by the same three investigators according to the Newcastle–Ottawa Scale (NOS) , recessive model (TT vs. CT+CC), and allelic model , respectively.All statistical analyses were conducted using Stata statistical software ver. 11.0  and Review Manager ver. 5.0 . All tests were two-sided and a 2 statistic  p<0.1 and the measure of I2 was >50%, indicating significant heterogeneity between studies, the ORs were pooled using a fixed effects Mantel–Haenszel method Heterogeneity between the studies was analyzed using the Cochran’s Q test and the ISensitivity analysis was conducted by limiting the meta-analysis to high quality studies (NOS score ≥8). We also performed the analyses a second time by limiting the studies according to the HWE and excluding those that included myocardial infarction, obesity, or diabetes mellitus in the cases or controls. Sensitivity analysis was performed to identify alterations in the overall significance of the estimate.GNB3 C825T polymorphism and hypertension, and to estimate the combined estimate over time Cumulative meta-analysis was performed to identify the influence of the first published study on the subsequent publications concerning the relationship between the Publication bias was assessed using the Egger's regression test and Begg's 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 The present study met the PRISMA statement requirements . ThroughGNB3 C825T polymorphism and hypertension. Among these articles, three publications GNB3 C825T polymorphism and ischemic stroke were identified while one Of the 66 studies, eight compared males and females to assess an association between the All of the included studies were case-controlled in design. The main characteristics of the included studies are summarized in GNB3 C825T polymorphism and hypertension or stroke were identified using the random effects model for I2>50%, which suggested significant heterogeneity. However, in most of the models, I2 was ≥70%, which indicated high heterogeneity GNB3 C825T polymorphism and the risk of hypertension was only detected in the allelic model . No evidence of significance was identified in the dominant model  or the recessive model . However, none of the comparison models found an association between the GNB3 C825T polymorphism and stroke  (GNB3 C825T polymorphism was not a risk factor for stroke.All models concerning the association of the ctively) . After eWe also performed a meta-analysis to detect any association between males and females; however, only the recessive model  identified a risk of hypertension among females.GNB3 C825T polymorphism and hypertension and triggered the identification of subsequent related studies that tried to replicate the initial results. In the allelic, dominant, and recessive models, the study by Benjafield et al. In the cumulative meta-analysis by year of publication, the ORs and 95% CIs became more stable . Study bTo further clarify heterogeneity among the studies, we performed subgroup analysis. Regarding the hypertension study population, the status of the HWE and the source of control had a critical role in heterogeneity . No statistical evidence of publication bias was identified regarding the GNB3 C825T polymorphism and its association with stroke.Funnel plots were constructed and the Egger's test was performed to assess publication bias of the studies. Funnel plots should be symmetrical when no publication bias exists  and 5. RGNB3 825T polymorphism was associated with an increased risk of hypertension, obesity, metabolic syndrome, atherosclerosis, and diabetes mellitus. Besides, the GNB3 825T allele was found to significantly increase the risk of clinical ischemic stroke in Caucasians, but not subclinical cerebral infarct GNB3 C825T polymorphism and essential hypertension or stroke.Stroke is a significant event that leads to increased mortality and morbidity and hypertensive individuals reportedly have a greater incidence of stroke than normotensive individuals. Genetic factors as well as obesity, high sodium intake, physical inactivity, low potassium diets, and alcohol consumption contribute to the occurrence of hypertension, and essential hypertension status may play a role in the etiology of stroke either through effects on blood pressure levels or through separate pathways GNB3 825T allele had a weak association with essential hypertension. However, after we restricted the studies according to the HWE and included only those without other diseases, such as diabetes and myocardial infarction, all of the compared models failed to identify an association between the GNB3 825T allele and hypertension. Similarly, when we performed sensitivity analysis with the inclusion criteria of “Asian” or “Caucasian,” no evidence of an association was obtained, which might be due to heterogeneity between the studies. Besides, the funnel plot was asymmetric in the recessive model for p = 0.043, so publication bias must also be considered. In addition, our results were consistent with those reported in previous studies GNB3 C825T polymorphism and essential hypertension in Africans.Overall, our meta-analytical results showed that the GNB3 C825T polymorphism was not associated with stroke. When we retrieved studies on ischemic stroke cases or limited the studies according to the HWE or an NOS score of ≥8, similar results were obtained, suggesting that our initial results were reliable and in line with most of the included studies. But, considering that most of the included stroke patients were Asian, our results cannot be directly used to extrapolate a correlation between the GNB3 c825T polymorphism and stroke in Caucasians, Africans, or other ethnicities.Interestingly, the p = 0.0001). This result was in agreement with a previous study p = 0.337). Therefore, it is likely that a higher T allele frequency is not necessarily indicative of an increased occurrence of hypertension.In addition, we tested the T allele frequency in controls (hypertensive population) , and fouGNB3 825T allele was only slightly associated with an increased risk of essential hypertension compared to non-carriers. But, the GNB3 C825T polymorphism failed to contribute to the risk of stroke, thus it was clear that the polymorphism contributed to hypertension and stroke differently. Therefore, gene-gene interactions should be taken into consideration. Until now, >500 candidate genes for hypertension have been suggested from a variety of genetic studies, and this number continues to increase GNB3 C825T polymorphism with hypertension or stroke is required. The interactions between environmental and genetic factors constitute a key issue in the pathogenesis of hypertension and stroke. Most of the susceptibility genes for common diseases such as hypertension do not have a strong primary etiological role in disease predisposition, but rather code response elements to exogenous environmental factors. Therefore, a genetic marker may have only a modest affect on calculating risk in individuals who minimize exposure to environmental factors, but a major effect in individuals exposed to high-risk environment factors GNB3 presents a number of functional alleles that influence hypertension susceptibility. Therefore, those populations that have a high prevalence of the GNB3 825T allele also have a higher prevalence of heat-adapted alleles at other SNPs. Physical inactivity, increased body mass, obesity, and smoking may also influence the risk of hypertension and stroke differently.Generally, the GNB3 TT genotype was marginally associated with hypertension among females, and no evidence of an association with hypertension was found in males. In the cumulative meta-analysis, three models showed evidence of a non-association between GNB3 alleles and hypertension or stroke. Two primary causes may account for this discrepancy. First, females have dominant parasympathetic and subordinate sympathetic activities compared to males, and, secondly, estrogen plays an important role in gender-related differences in the autonomic nervous system GNB3 825T allele with hypertension.In addition to race, gender also seems to be an important risk factor for adverse cardiovascular events, such as hypertension and stroke. Suwazono et al. GNB3 C825T polymorphism. Thus, more population-based studies with large sample sizes are required. Despite these limitations, this meta-analysis was designed to overcome the limitations of individual studies, thus the results should be more reliable. Since the GNB3 C825T polymorphism appears to be a useful marker to predict the relative risk of diseases, such as hypertension and stroke, this meta-analysis is better suited in a preventive aspect to identify certain genotypes that will be most likely to benefit from pharmacological interventions.Some limitations of the present meta-analysis should be considered. Firstly, all of the included studies mostly involved Caucasians and Asians, thus studies on other ethnic populations are needed. Secondly, all of the included studies were case-controlled and all of the cases involved survivors of hypertension and stroke. Finally, the number of stroke cases were limited and had relatively weak statistical power to detect potential risks of the GNB3 825T allele may be a good indicator of hypertension; however, it had no association with hypertension in Asians and Caucasians and there was lack of evidence to support an association with stroke in Asians. Therefore, multiethnic studies with much larger sample-sizes are required to better evaluate the association between the GNB3 C825T polymorphism and hypertension or stroke.In summary, the overall analysis of available evidence suggested that the Appendix S1PRISMA Checklist.(DOC)Click here for additional data file.


Numerous studies have evaluated the association between regulated upon activation, normal T cells expressed and secreted (RANTES) gene polymorphisms (−403G/A and −28C/G) and risk of pediatric asthma. However, the results have been inconsistent. A meta-analysis of the association between RANTES gene polymorphisms and pediatric asthma risk was performed in the current study. A search for published literature was conducted in the Google Scholar, PubMed and the CNKI databases (January 2000 to April 2012) and seven studies were retrieved. The associations between RANTES gene polymorphisms and pediatric asthma risk were estimated by pooled odds ratio (OR) and 95% confidence interval (CI) using a fixed- or random-effects model. Meta-analysis results revealed no significant association between the −403G/A polymorphism and risk of pediatric asthma. In the subgroup analysis by ethnicity, no association was identified between the −403G/A polymorphism and pediatric asthma risk in Caucasian and Asian populations. In the −28C/G group, the meta-analysis indicated a significant association between the −28C/G polymorphism and pediatric asthma susceptibility among the total population . However, when the subgroup analysis was performed by ethnicity, no significant associations were identified in Asians and Europeans. This result suggests that the −28C/G polymorphism may not be associated with pediatric asthma risk, while the observed increase in the risk of pediatric asthma may be due to racial differences. Additional large-scale studies are required to provide conclusive evidence on the effects of RANTES gene polymorphisms on the risk of pediatric asthma. Asthma is a chronic inflammatory disease of the respiratory airways leading to episodes of wheezing, shortness of breath, chest tightness and coughing . ApproxiThe regulated upon activation, normal T cells expressed and secreted (RANTES) protein is one of the most extensively studied chemokines in allergic and infectious diseases . RANTES The data were independently gathered in duplicate by two investigators on the basis of a standard protocol (Y.M.L. and L.F.C.). Discrepancies between the investigators were settled by discussion until consensus was reached. Literature databases were searched, including PubMed, Google Scholar and the China National Knowledge Infrastructure (CNKI) databases. The following key words and subject terms were searched: ‘RANTES’, ‘−403G/A’, ‘−28C/G’, ‘pediatric asthma’ and ‘gene polymorphism’. The reference lists of retrieved reviews and articles were hand-searched. The search was without restriction on language and was conducted on human subjects. The literature search was updated on 30 May 2012.The titles and abstracts of all citations identified by the literature search were reviewed. Selection criteria were then applied to all potentially relevant studies. The selection criteria for inclusion in the meta-analysis were: i) case-control studies conducted to evaluate the association between RANTES gene polymorphisms (−403G/A and −28C/G) and pediatric asthma risk; ii) sufficient genotype data were presented to calculate the odds ratios (ORs) and 95% confidence intervals (CIs); iii) the study should clearly describe pediatric asthma diagnoses and the sources of cases and controls. Studies were excluded when: i) they were non-case-control studies that evaluated the association between RANTES gene polymorphisms and pediatric asthma risk; ii) they were case reports, letters, reviews or editorial articles; iii) they were studies based on incomplete raw data and no usable data were reported; iv) they contained duplicate data.The following characteristics were collected from each study: first author, year of publication, region of the first or corresponding author, ethnicity, number of cases and controls, number of genotypes and evidence of Hardy-Weinberg equilibrium HWE; . Ethnici2 test. The effect of heterogeneity by the Q-test and I2 test was also quantified. I2 ranges between 0 and 100% and I2 values of 25, 50 and 75% were defined as low, moderate and high estimates, respectively. When a significant Q-test (P<0.10) or I2>50% indicated heterogeneity across studies, the random-effects model was used for meta-analysis, otherwise the fixed-effects model was calculated. Sensitivity analysis was performed to assess the stability of the results by excluding the study by Sohn et al . The OR corresponding to the 95% CI was used to assess the intensity of the association between RANTES gene polymorphisms (−403G/A and −28C/G) and pediatric asthma under a homozygote comparison (AA vs. aa), a heterozygote comparison (AA vs. Aa), a dominant model and a recessive mode between groups. In the current study, the dominant model was defined as Aa+aa vs. AA, where ‘A’ and ‘a’ are major and minor alleles, respectively, and the recessive model as aa vs. AA+Aa. The distribution of genotypes in the included studies was tested for HWE using the χhn et al  with genet al  and pediatric asthma risk is shown in et al  with genet al . The difAsthma is the most common chronic childhood disease . The patet al  and susceptibility to pediatric asthma. Additonally, the results of our meta-analysis did not show any significant association between −403G/A polymorphism and pediatric asthma risk. As for −28C/G, the meta-analysis indicated a significant association between −28C/G polymorphism and pediatric asthma susceptibility among the total population . However, when the subgroup analyses were performed by ethnicity, no significant associations were identified in Asian and European populations. This result suggests that the −28C/G polymorphism may not be associated with pediatric asthma risk and the observed increase in the risk of pediatric asthma may be due to a bias of racial differences. Sensitivity analysis was performed to assess the stability of the results by excluding the study by Sohn et al  with genThere were certain limitations to this meta-analysis. First, the linkage disequilibrium of −403G/A and −28C/G of the RANTES gene may synergistically increase the risk of asthma . There wIn conclusion, these results suggest that RANTES gene polymorphisms (−403G/A and −28C/G) may not be involved with the susceptibility of pediatric asthma. Owing to the previously mentioned limitations, the findings should be verified by further research in the near future.


The Ser326Cys polymorphism in the human 8-oxogunaine glycosylase (hOGG1) gene with lung cancer susceptibility had been investigated, but results were inconsistent and underpowered. The aim of this study was to conduct a meta-analysis assessing the association of hOGG1 Ser326Cys polymorphism with risk of lung cancer.Relevant studies were identified through a search of MEDLINE, PubMed, Web of Science, EMBASE, and Chinese Biomedical Literature database (CBM) using terms “lung cancer”, “hOGG1” or “OGG1”, “polymorphism” or “variation” and the last search updated on May 1, 2013. In this meta-analysis, we assessed 30 published studies involving 22,475 subjects that investigated the association between the hOGG1 Ser326Cys polymorphism and lung cancer susceptibility.heterogeneity = 0.000), and recessive model: OR = 0.543; 95% CI = 0.399–0.739; Pheterogeneity = 0.000). Similarly, in the stratified analyses by ethnicity, significantly increased risks were found among Asians for homozygote comparison , and dominant model , and Caucasians for dominant model , and recessive model . In population-based populations, marginally significant increased risks were found in dominant model  and recessive model . We also found a significant difference between hOGG1 Ser326Cys genotype and lung cancer susceptibility in studies with hospital-based controls for homozygote model ,dominant model .Overall, the hOGG1 Ser326Cys polymorphism was not associated with lung cancer susceptibility in different genetic models (dominant model comparison: OR = 0.133; 95% CI = 0.111–0.161; POur data showed that the hOGG1 Ser326Cys polymorphism contributed to the risk of lung cancer.http://www.diagnosticpathology.diagnomx.eu/vs/3842531131031605The virtual slides for this article can be found here:  Lung cancer has become one of the most common malignancies all over the world with an extremely low survival rate is one of the leading causes to contribut to cancer mortality . A numbeThe 8-oxoguanine lesion is one of themajor forms of oxidative DNA damage ,13, and A comprehensive search strategy was conducted towards the electronic databases including MEDLINE, PubMed, Web of Science, EMBASE, and Chinese Biomedical Literature database (CBM) using terms “lung cancer”, “hOGG1” or “OGG1”, “polymorphism” or “variation” and the last search updated on May 1, 2013. Among the studies retrieved, eligible ones were determined and their bibliographies were evaluated for other relevant publications. Review articles and bibliographies of other relevant studies identified were hand-searched to identify additional eligible studies. Only published studies with full text articles were included. When more than one of the same patient population was included in several publications, only the most recent or complete study was used in this meta-analysis.The following inclusion criteria were used to select literatures for the meta-analysis: (1) Only the case–control studies were considered; (2) The paper should clearly describe lung cancer diagnoses and the sources of cases and controls; (3) The authors must offer the size of the sample, OR and their 95% CI or the information that can help infer the results in the papers . The exclusion criteria were: (1) none-case–control studies; (2) control population including malignant tumor patients; and (3) duplicated publications.Information was carefully extracted from all eligible publications independently by two authors according to the inclusion and exclusion criteria listed above. An agreement was reached by discussion between the two reviewers whenever there was a conflict. The following data were collected from each study: first author’s surname, year of publication, country, ethnicity, source of controls , and numbers of cases and controls with the Ser/Ser, Ser/Cys, and Cys/Cys genotypes, respectively. Different ethnicity descents were categorized as Caucasian and Asian population. When studies included subjects of more than one ethnicity and were able to separate, data were extracted separately for each ethnic group. We did not define any minimum number of patients to include a study in our meta-analysis.Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association between the hOGG1 Ser326Cys polymorphism and lung cancer risk according to the method of Woolf . HomozygHeterogeneity assumption was checked by the Chi-square-based Q-test . A P-valStudies relevant to the searching words were retrieved originally. The initial search algorithm retrieved 229 references. After careful review of the abstracts, 155 of studies were excluded because they obviously did not meet the inclusion criteria for the meta-analysis. After this exclusion, 74 studies were left for full publication review. After review of the complete articles, 47 studies were excluded because of a lack of sufficient information or methods discrepancies. A total of 30 studies involving 10,327 lung cancer cases and 12,148 controls were ultimately analyzed   , heterozygote model , and also the Ser-allele were not associated with an increased cancer risk compared with the Cys-allele model .Table ) Figure , and rec) Figure  when allheterogeneity = 0.064, P = 0.032), dominant model  (Table heterogeneity = 0.015) (Table heterogeneity = 0.015) , dominant model , and recessive model   and recessive model  , heterozygote comparison (P = 0.023 for Ser/Ser vs Ser/Cys model and P = 0.006 for Ser/Cys vs Cys/Cys), dominant model comparison (P = 0.000), recessive model comparison (P = 0.000) and Ser-allele vs Cys-allele comparison (P = 0.000). After assessing the source of heterogeneity for all genetic model comparison by subgroup analysis on ethnicity and control sources, the heterogeneity was partly decreased. When we deleted these three studies ,37,44 foA single study involved in the meta-analysis was deleted each time to reflect the influence of the individual data set to the pooled ORs, and the corresponding pooled ORs indicated that three studies were the main origin of heterogeneity. The heterogeneity was completely removed after exclusion of these studies. Although these studies did not follow HWE, the corresponding pooled ORs were not materially altered with or without including them almost in all genetic models. Similarly, no other single study influenced the pooled OR qualitatively, as indicated by sensitivity analysis, suggesting that the results of this meta-analysis are stable (Data were not show in this paper).Begg’s funnel plot and Egger’s test were performed to assess the publication bias of the literature. The shapes of the funnel plots did not reveal any evidence of obvious asymmetry in all comparison models Figure . FurtherIt is well recognized that there is a range of individual susceptibility to the same kind of cancer even with identical environmental exposure. Host factors, including polymorphisms of genes involved in carcinogenesis, may have accounted for this difference. Therefore, genetic susceptibility to cancer has been a research focus in scientific community. Among the common single nucleotide polymorphisms (SNPs) of the hOGG1 gene, one located in exon 7, resulting in an amino acid substitution of serine (Ser) with cysteine (Cys) at codon 326 , has been demonstrated to affect hOGG1 function . The proThe meta-analysis of Hung et al. showed that the summary OR was 1.37 (95% CI = 1.02–1.82) for the Cys/Cys genotype in various ethnic populations combined . Li et aHeterogeneity is a potential problem when interpreting the results of all meta-analyses. As looked through our study carefully, we found that the three studies ,37,45 weSome limitations of this meta-analysis should be addressed. Firstly, lung cancer is a multi-factorial disease that results from complex interactions between many genetic and environmental factors. This means that there will not be single gene or single environmental factor that has large effects on lung cancer susceptibility. Our results were based on unadjusted estimates, while a more precise analysis should be conducted if individual data were available, which would allow for the adjustment by other covariates including age, sex, family history, environmental factors and lifestyle. Secondly, in the subgroup analyses by ethnicity, control sources, the number of subjects was relatively small, not having enough statistical power to explore the real association. Thirdly, the controls were not uniformly defined. Although most of the controls were selected mainly from healthy populations, some had respiratory disease. Therefore, non-differential misclassification bias was possible because these studies may have included the control groups who had different risks of developing lung cancer.Despite some limitations listed above, this work suggests that the hOGG1 Ser326Cys variant is a risk factor for developing lung cancer. Additionally, we found that this phenomenon was more prominent in subgroups such as in Asians. However, it is necessary to conduct large studies using standardized unbiased methods, homogeneous lung cancer patients and well matched controls, with the assessors blinded to the data. Moreover, gene–gene and gene–environment interactions should also be considered in future analysis. Such studies taking these factors into account may eventually lead to our better, comprehensive understanding of the association between the hOGG1 Ser326Cys polymorphism and lung cancer risk.The authors declare that they have no competing interests.ZX, LY, and XZ carried out the meta-analysis study, drafted the manuscript and involved in revising the manuscript critically for important intellectual content. ZX participated in the design of the study and revised the manuscript. ZX, LY, and XZ carried out the meta-analysis study and drafted the manuscript. ZX, LY, and XZ participated in the design of the study, drafted the manuscript and revised the manuscript. All authors read and approve the final manuscript.


ALDH2*1/*2 and less-active ADH1B*1/*1, and smoking, are risk factors for esophageal SCC. We previously examined telomere shortening in the esophageal epithelium of alcoholics, and suggested a high prevalence of chromosomal instability in such individuals. In the present study, we attempted to analyze telomere lengths in 52 DIULs with reference to both their size and multiplicity, ALDH2 and ADH1B genotypes, and smoking history. Patients with DIULs <10 mm (n = 42) had significantly longer telomeres than those with DIULs ≥10 mm . No significant differences in telomere length were recognized between the ALDH2 and ADH1B genotypes  or among four groups of patients divided according to smoking history . Patients without multiple DIULs (n = 17) had significantly longer telomeres than patients with multiple DIULs . It is suggested that alcoholism reduces telomere length in the esophagus, irrespective of genotype or smoking habit. Telomere shortening may not generate cancer directly, but may create conditions under which SCC can develop more easily, depending on subsequent exposure to carcinogens.Chromoendoscopy with Lugol iodine staining provides important information on the development of squamous cell carcinoma (SCC). In particular, distinct iodine-unstained lesions (DIULs) larger than 10 mm show a high prevalence in high-grade intraepithelial neoplasia. It has also been reported that inactive  Squamous cell carcinoma (SCC) is still the predominant histologic type of esophageal cancer in areas where there is a high risk of developing it. Turkish, Mongolian, and African-American populations have a risk of developing esophageal cancer two to three times higher than that of American Caucasians, regardless of sex Telomeres are repetitive G-rich DNA sequences and associated binding proteins found at the ends of linear eukaryotic chromosomes, and appear to play a key role in preventing genomic instability in situ (CIS) have been shown to be significantly shorter than those in age-matched controls. These results have confirmed that our method for telomere measurement is accurate and reproducible.Using Southern blotting and quantitative fluorescence in situ hybridization (Q-FISH), we have demonstrated that telomere shortening occurs in almost all human organs and tissues, including esophageal epithelium, with aging ALDH2*1/*2 and ADH1B*1/*1 genotypes For early diagnosis of esophageal neoplastic lesions, chromoendoscopy with iodine staining is an important tool ALDH2*1/*2 and less-active ADH1B*1/*1 genotypes, smoking, and frequent drinking of strong alcoholic beverages straight ALDH2*1/*2 and ADH1B*1/*1 genotypes are very rare among Caucasians, they account for 13.0% of alcoholics in Japan SCC of the esophagus is quite rare in young people ALDH2*1/*2 and/or less-active ADH1B*1/*1 genotype, and smoking, are risk factors associated with telomere shortening in alcoholics.In the present study, we analyzed the telomere lengths of DIULs, and examined the relationship between DIUL telomere length and DIUL size or multiplicity. Also, we attempted to clarify whether the inactive AY was responsible for aquiring informed consent from the patients included in this study, and the participants provided written informed consent to participate in this study. The ethics committees of Kurihama Medical and Addiction Center and Tokyo Metropolitan Institute of Gerontology approved the consent procedure. All specimens used for this study were obtained from Japanese alcoholic patients who underwent upper gastrointestinal endoscopy at Kurihama Medical and Addiction Center. No inclusion criteria other than being a Japanese alcoholic patient were considered for this study. No information on any of the samples studied was able to identify any of the individual patients included.Endoscopic examination and biopsy were performed by one (AY) of the authors (2008–2011) at the National Hospital Organization Kurihama Medical and Addiction Center. All of the alcoholics who participated in this study met the DSM-IV criteria for alcohol dependence stipulated by the American Psychiatric Association Biopsy specimens were fixed for 5 hours in 10% buffered formalin, and then subjected to standard tissue processing and paraffin embedding. Tissues were sliced serially into sections 3 µm thick for hematoxylin and eosin (HE) staining, and into sections 2 µm thick for Q-FISH.Histologic examinations of the specimens were performed by 4 pathologists , who were experts in gastrointestinal pathology. Inter-observer variations among histological diagnoses were discussed among these observers using a multi-headed teaching microscope, and a consensus was reached.Any biopsy specimens with histologically evident atypia, dysplasia, or malignancy were excluded. We used the telomere length of basal cells as the representative value for each specimen, because tissue stem cells are considered to reside in the basal layer PCR-restriction fragment length polymorphism was employed to determine the ALDH2 and ADH1B genotypes using DNA samples from blood cells. The samples for this purpose were obtained with informed consent from patients admitted to the National Hospital Organization Kurihama Alcoholism Center, and the data obtained were not released until completion of all the telomere analyses.The slides were processed by the FISH method, as reported previously 5′-CCCTAACCCTAACCCTAA-3′; catalogue number F1002, Fasmac, Japan) and the centromere (Cenp1-fluorescein isothiocyanate (FITC) probe: 5′-CTTCGTTGGAAACGGGGT-3′; custom-made, Fasmac), and the nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI) .Tissue sections were hybridized with peptide nucleic acid (PNA) probes for the telomere  camera  mounted on an epifluorescence microscope  equipped with a triple band-pass filter set for DAPI/FITC/Cy3  and a x40 objective lens . Microscope control and image acquisition were performed using the Image-Pro Plus software package . The captured images were analyzed using our own tissue analysis software, ‘TissueTelo Ver. 3.1’, which estimates the telomere to centromere ratio (TCR) and the normalized TCR (NTCR) of individual nuclei, as reported previously As a control for variations in sample preparation, we also performed Q-FISH on a cell-block section from a cultured cell strain, TIG-1 In order to analyze the independence of chromoendoscopy findings and ALDH2 and ADH1B genotypes, chi-square test was performed among ALDH2 and ADH1B genotypes and the size or multiplicity of DIULs.p<0.05 were considered to be significant.The distributions of NTCRs in each case did not show a normal distribution The 52 cases analyzed were divided into two groups on the basis of the size of the DIUL from which the biopsy specimen had been taken: <10 mm (42 cases) and ≥10 mm (10 cases). Thirty-five of the 52 cases had multiple DIULs and 17 cases did not have multiple DIULs. Finally, 52 cases without dysplasia  . The median NTCR showed no significant inter-group difference (p = 0.0581)  .p = 0.0403, and 0.0275, respectively)  .For the combination of size and multiplicity (present or absent), we separated the cases into 4 groups , and compared the NTCR among them. Although no significant difference was found, the SA group had a larger NTCR than the SP group (Table S1).p = 0.5630, 0.4499, respectively) (There were no significant differences in mean and median NTCRs between the groups divided according to active or inactive ALDH2 genotype (ctively) .p = 0.7837, 0.7715, respectively) (There were no significant differences in mean and median NTCRs between the groups divided according to active or less-active ADH1B genotype (ctively) .For the combination of ALDH2 and ADH1B genotype, we separated all the cases into 4 groups , and compared among them. However, no significant differences in NTCR were evident among these 4 groups (Table S2).p = 0.9561) (There were no significant differences in mean and median NTCRs among the groups categorized according to smoking history ( 0.9561) .p = 0.117), but the ADH1B genotype did show such an association . Both the ALDH2 and ADH1B genotypes were also associated with the presence of multiple DIULs .The ALDH2 genotype showed no association with the presence of DIULs ≥10 mm in diameter  Telomeres were shorter in esophageal epithelium associated with DIULs ≥10 mm in diameter or multiple DIULs. (B) In DIULs of alcoholics, telomere lengths showed no significant differences between the ALDH2 and ADH1B genotypes, or between individuals with different smoking histories.The International Agency for Research on Cancer of the World Health Organization has classified ethanol and acetaldehyde, which are associated with alcoholic beverages, as Group 1 human carcinogens The epithelial cells of DIULs ≥10 mm in diameter had shorter telomeres than those within smaller DIULs. When DIULs become larger, their epithelial telomeres might become shorter, and thus the epithelium in larger DIULs may have greater chromosomal instability than that in smaller DIULs, being compatible with the fact that SCC including CIS occurs more frequently in larger than in smaller DIULs Pavanello and colleagues per se rather than the causative impact of ALDH2 and ADH1B genotype on the development of DIULs.Alteration of p53 tumor suppressor protein is suspected to be the key molecular event in multifocal carcinogenesis in the esophagus, head and neck, and very high levels of p53 protein accumulation have been shown to occur in early esophageal SCC in Japanese alcoholic men In any event, telomere shortening may not cause cancer directly, but may create conditions in which SCC can develop more easily, if subsequent exposure to carcinogens occurs.Our findings indicate that in DIULs of the esophagus in alcoholics, telomere lengths in epithelia do not differ significantly among individuals with different ALDH2 or ADH1B genotypes. Chromoendoscopy with iodine staining appears to provide evidence of telomere shortening in the esophageal epithelium.Table S1Combination of Diameter and Multiplicity of DIULs in NTCRs.(DOCX)Click here for additional data file.Table S2Combination of ALDH2 and ADH1B genotypes in NTCRs.(DOCX)Click here for additional data file.