Type 2 Diabetes (T2D) and other chronic diseases are caused by a complex combination of many genetic and environmental factors. Few methods are available to comprehensively associate specific physical environmental factors with disease. We conducted a pilot Environmental-Wide Association Study (EWAS), in which epidemiological data are comprehensively and systematically interpreted in a manner analogous to a Genome Wide Association Study (GWAS).We performed multiple cross-sectional analyses associating 266 unique environmental factors with clinical status for T2D defined by fasting blood sugar (FBG) concentration ≥126 mg/dL. We utilized available Centers for Disease Control (CDC) National Health and Nutrition Examination Survey (NHANES) cohorts from years 1999 to 2006. Within cohort sample numbers ranged from 503 to 3,318. Logistic regression models were adjusted for age, sex, body mass index (BMI), ethnicity, and an estimate of socioeconomic status (SES). As in GWAS, multiple comparisons were controlled and significant findings were validated with other cohorts. We discovered significant associations for the pesticide-derivative heptachlor epoxide , and the vitamin γ-tocopherol . Higher concentrations of polychlorinated biphenyls (PCBs) such as PCB170  were also found. Protective factors associated with T2D included β-carotenes .Despite difficulty in ascertaining causality, the potential for novel factors of large effect associated with T2D justify the use of EWAS to create hypotheses regarding the broad contribution of the environment to disease. Even in this study based on prior collected epidemiological measures, environmental factors can be found with effect sizes comparable to the best loci yet found by GWAS. It is becoming clear that most non-communicable diseases are a result of a complex combination of genetic processes and the environment Perhaps the lack of impact of GWAS comes from not comprehensively considering environmental factors in disease. T2D provides an specific example: while genetics play a large role The current paradigm to search for the effects of multiple environmental chemicals utilizes molecular tools and model systems The EWAS consists of two methodological steps that have analogs in a GWAS. First, we consider a panel of 266 unique environmental assays, or environmental “loci”, measured across cases of diabetics and controls, yielding several environmental factors with significantly high association with T2D while controlling for multiple hypotheses. Second, we validate the associations by taking advantage of data from other cohorts in NHANES. With EWAS, we are able to hypothesize about new associations with T2D and reconfirm others. The results from EWAS can better inform about environmental factors that need to be measured in genetic studies to begin to provide us insight in regards to disease etiology.The NHANES is a publicly available dataset made available by the CDC and National Centers for Health Statistics and all participants have provided written consent.LBXVIE from 1999–2000 described “A-Tocopherol ug/dL” and variable with symbol LBXATC from 2001–2002 also described “a-tocopherol ug/dL”, we harmonized onto the single symbol for both, LBXATC.We associate 266 unique environmental factors with T2D status from the NHANES. We downloaded the all of the available NHANES data for 1999–2000, 2001–2002, 2003–2004, and 2005–2006 cohorts and collated corresponding variables across them. For example, if a variable with symbol There was a total of 543 factors in our EWAS, but not all factors were present in all cohorts: 111 factors measured in the 1999–2000 cohort, 146 from 2001–2002, 211 from 2003–2004, and 75 from 2005–2006. This comprised of 266 unique environmental factors in total, with 157 factors measured in more than one cohort. Using NHANES categorization, we binned factors into 21 “class” groupings in order to discern patterns among related groups of factors, analogous to chromosomal units in GWAS . DiffereTrichomonas vaginalis, an infectious pathogen found primarily in women.We omitted from our EWAS 73 factors that varied little across individuals in our sample. Specifically, we omitted those that had a majority (>90%) of the observations below a detection limit threshold as defined by in the NHANES codebook. We also removed factors that targeted a subset of the population, such as the test for T2D cases were individuals who had a fasting blood glucose (FBG) level greater or equal to 126 mg/dL, as advised by the American Diabetes Association (ADA) R with the survey module to conduct all survey-weighted analyses, and replicated our results with the STATA program We used survey-weighted logistic regression to associate each of the 543 environmental attributes with diabetes status while adjusting for age, sex, body mass index (BMI), ethnicity, and an estimate for SES . We acknExposures were captured either as continuous or a categorical variable. Most chemical exposure data arising from mass spectrometry or absorption measurements occurred within a very small range and had a right skew; thus, we log transformed these variables. Further, we applied a z-score transformation  in order to compare odds ratios from the many regressions. Similarly, for categorical variables, we made the definition of the referent consistent, defining them to be the “negative” results of the test.We calculated the false discovery rate (FDR), the estimated proportion of false discoveries made versus the number of real discoveries made at a given significance level, to control for type I error due to multiple hypotheses testing in associating the factors to disease status To improve our power, we used the four independent cohorts to validate significant findings . We consWe fit a final logistic regression model with data combined from multiple NHANES cohorts utilizing all measurements for a specific environmental factor, attaining an overall odds ratio. The covariates of the final model were age, sex, BMI, ethnicity, SES, and cohort. We computed new sample weights for the combined datasets by taking the average of the original sample weights as described by the NHANES analytic guidelines We conducted 3 secondary analytic tests for the validity and sensitivity of our final estimates. We first attempted to check for reverse causality, or association of exposure due to T2D diagnosis. Our second test attempted to take into account the lipophilic characteristics of the environmental factors found. Our last test attempted to take into account recent food and supplement consumption as a potential bias for exposure measures. For adequate sample size and ease of comparison to the final fit model, we utilized all available data combining multiple NHANES cohorts as the sample to conduct these three tests, described below.To attempt to account for reverse causality, we recomputed our models omitting those individuals who had been diagnosed with diabetes, defined here as those answering yes answers submitted on a NHANES health questionnaire (“Doctor told you have diabetes?”). We then refit our final models with individuals only showing biochemical evidence of T2D without actual diagnosis.Our second test attempted to account for the lipophilic chemical characteristics of our significant factors. Many of the environmental factors measured in NHANES absorb readily in fatty tissue; presence of fatty tissue is also associated with T2D and a potential confounder. Thus, we recomputed the models taking into account total triglycerides and cholesterol measured in blood specimen of participants.In our third test, we attempted to compare dietary and supplement consumption of cases or controls gathered from 24- and 48-hour recall and supplement use questionnaires reasoning that recent intake may confound exposure-disease association. The NHANES data contains amount of food components consumed based on the dietary recall available for all participants examined above. Specifically, amounts of food components are computed from the questionnaire using the United States Department of Agriculture (USDA) Food and Nutrient Database. Some of the vitamin and nutrient components included vitamin A, vitamin B-6, vitamin B-12, vitamin C, vitamin E, vitamin K, carotenes, lycopene, thiamin, riboflavin, niacin, folate, calcium, iron, magnesium, phosphorus, potassium, sodium, iron, zinc, copper, and selenium. Other components included macronutrients, such as protein, carbohydrates, fat, fiber, and cholesterol. The total amount of food components considered numbered 51 to 63 for the different cohorts. Further, the 2003–2004 and 2005–2006 cohorts contained both 24- and 48-hour recall data. Supplement use included count of consumption of vitamins, minerals, botanicals, and/or their mixture of them over the past month prior to the survey. To check for possible confounding by recent consumption, we added each food and supplement variable to the logistic regression models specified above and re-evaluated significance and effect size of the validated environmental factors. We coded food component content as the logarithm (base 10) of the amount entered. We coded supplement use as an integer count value. We acknowledge the potential of bias with the use of questionnaire data and a pre-determined database of food items but assumed it was a reliable proxy of consumption and behavioral data in lieu of other information.Using GWAS as inspiration, we systematically and comprehensively assessed the association of 266 unique environmental factors measured in the NHANES with T2D. Further, we validated these associations by observing the significance of factors in other NHANES cohorts.2 test) except for 2005–2006. Furthermore, there was a significant association between lowest SES (first tertile of poverty index) and T2D  in for the 1999–2000, 2001–2002, and 2005–2006 cohorts respectively. While we did not see a univariate association between ethnicity and T2D as diagnosed by FBG, we did confirm previously reported associations of ethnicity with T2D when stratifying by age and sex Several vitamins were found to have levels inversely associated with T2D. The first type included an antioxidant in the isoforms of β-carotene  of 0.6; 95% confidence interval (CI) 0.5–0.7; p<0.001). For the prototypical participant, high levels of trans or cis β-carotene equated to a 9% improvement in risk (15 vs. 6%) for T2D status. We were able to confirm the inverse association of β-carotenes seen in multiple epidemiological studies in Saudi Arabia We discovered a vitamin that increased risk for T2D. Surprisingly, γ-tocopherol, a form of vitamin E, was highly significantly and positively associated with T2D  in two cohorts  and nearly significant in the two others . For the prototypical participant, low levels of the γ-tocopherol equated to a 7% improvement in risk (13% vs. 6%). To our knowledge, this is a novel association between γ-tocopherol and T2D.We found organochlorinated pesticides and polychlorinated biphenyls (PCBs), both related pollutant factors, to be a highly positively associated with T2D. Among the PCBs, we specifically discovered PCB170 . The effect sizes in the individual cohorts for PCB170 were large . The models predicted up to 15% T2D risk for the prototype participant, more than double the risk of those with low concentrations of PCB170. The association between the class of PCBs with T2D has been well described within Native American Heptachlor epoxide, an oxidation product of the organochlorine pesticide heptachlor, was among the most highly associated factor  in our EWAS. The effect sizes in the individual cohorts were also large . The predicted probability for the prototypical participant with high levels of the pollutant was 7%, more than 2 times greater than those who had lower levels of this pollutant.We then attempted to test the validity of our final estimates by conducting 3 additional analytic tests. In the first test, we attempted to consider the possibility of “reverse causality” or differential exposure status due to T2D diagnosis. Second, we attempted to assess the effect of potential confounding bias due to the lipophilic characteristics on our final environmental factor effect estimates. Third, we attempted to assess the effect of recent nutrient and supplement consumption on our final effect estimates.To consider T2D diagnosis as a modulator of exposure, we removed all individuals who answered yes when questioned about a past history of diabetes in the NHANES health questionnaire (“Doctor told you have diabetes?”). We then recomputed the effect of exposure, adjusted for age, sex, SES, ethnicity, BMI, and cohort using the remaining individuals who showed biochemical evidence of T2D, but not carrying a diagnosis of T2D . For allWe next attempted to account for potential confounding bias of lipid levels. To assess the degree of possible confounding we refit the logistic regression adjusting for the logarithm (base 10) of total triglyceride and cholesterol levels in addition to age, sex, BMI, SES, ethnicity, and cohort . We did We then searched for differences in food and supplement consumption patterns between diabetics and non-diabetics for all 4 cohorts close to the time of survey derived from dietary recall and supplement use questionnaires. In comparing dietary nutrients, we did not observe a difference for any dietary nutrient except one between cases and controls. This exception included a lower total carbohydrate intake for diabetics versus controls, confirming that many diabetics may have known about their disease; specifically, the adjusted OR was 0.7  for a 10% increase in total carbohydrate consumption, adjusted for sex, age, ethnicity, SES, and cohort. We also observed an inverse association between any supplement use and T2D, with an adjusted OR of 0.6 , also consistent with our expectation of increased health awareness for those with T2D. However, we specifically could find no difference in consumption of carotenes or tocopherol (p = 0.85 and 0.2 respectively) between cases and controls, two of the validated nutrient factors found in our EWAS .Having observed some difference in consumption behavior between cases and controls, we then attempted to assess the influence of recalled dietary consumption on the environmental associations by recomputing the logistic regression models in presence of dietary and supplement use variables. Adding the new dietary or supplementary vitamin consumption variables did not attenuate the odds ratios (maximum change of 1–2%), nor did they lessen the strength of the associations for all of the 5 validated environmental factors described in We took a further step in assessing the strength of the environmental associations, adjusting for total triglycerides and cholesterol, any supplement use, and food intake simultaneously . SpecifiTo summarize of our secondary tests for validity, we concluded that reverse causality, recent food and supplement consumption, and total lipid levels did not substantially bias our effect estimates for the 5 validated factors. These tests were made possible by the extensive list of co-variates available in the NHANES.We have described a prototype Environmental-Wide Association Study (EWAS) and applied it to the study of Type 2 Diabetes (T2D), and validated many of our significant findings across independent cohorts and confirmed some of them through the literature. This pilot study is made possible by the examination of multiple cohorts present in the nationally representative NHANES dataset. We have rediscovered factors such as carotenes and PCBs with previously known association with T2D. Unexpectedly, we found higher levels of γ-tocopherol were associated with higher likelihood of T2D, independent of dietary intake. Of the components of Vitamin E, γ-tocopherol is the most abundant form in the US diet Another novel finding was in the significant association between heptachlor epoxide levels and T2D. Heptachlor is a pesticide; most uses of heptachlor were discontinued in the late 1980s While this study successfully demonstrates a prototype EWAS for T2D, this methodology can be reconfigured to measure the relationship between environmental factors and other disorders, such as obesity, lipid level abnormalities, hypertension, and/or cardiovascular disease. Methodologically, the EWAS takes inspiration from GWAS, which have been used to assess the correlation between genome-wide variability and disease.Like GWAS, the utility of EWAS lies in two types of hypothesis generation. First, the EWAS framework can be used to propose targets for further study. For example, many factors are correlated; some are similar structurally, such as the isomers of β-carotene, or co-occur in the environment, such as the PCBs and organochlorine pesticides. As we extend the GWAS analogy, these and other environmental factors could be said to be in “linkage disequilibrium” with each other. Just as is done for preliminary GWAS findings, EWAS findings can and should be used to identify further factors that may be in “disequilibrium,” for further detailed measurement and causal identification.We acknowledge that the measurement of 266 environmental factors is hardly a comprehensive study of the environment, but this is still a greater number of factors measured than the 30 microsatellite markers Another type of hypothesis we may generate is regarding the complex cause of disease. For example, we can now use an EWAS to hypothesize about “gene-environment” interactions and their relation to disease etiology. A future study addressing gene-environment interactions might be designed as a combination of both a GWAS and EWAS, where genetic variability is assessed simultaneously along with key environmental factors. While marginally more resource intensive, this type of study design could perhaps facilitate an explanation of disease causation that has eluded genomic-wide scans in addition and provide more accurate estimates of attributable risk.The EWAS allows for comprehensive and systematic analysis of the effects of the environment in association with disease on a broad scale. While many investigators have already utilized the NHANES to address the effect of a limited number of factors on disease, they do not provide a global view of these associations Limitations of this study remind us that measuring environment-wide aspects in relation to phenotypic states such as disease will be a difficult undertaking TCF7L2 gene While GWAS has allowed us to find novel variants associated with T2D of possible mechanistic importance and provided a model for a comprehensive study of the environment described here, associated variants have had only moderate effect sizes to date. Most of the risk loci identified with GWAS have small individual odds ratios, generally less than 1.3 FTO with T2D was nullified by accounting for BMI However, the correlated and dynamic nature of a multitude of environmental factors will hinder causal inference to a greater degree than GWAS It is imperative not only for epidemiologists and geneticists but also physicians and their patients to understand how multiple environmental factors may influence disease in a systematic fashion. Individuals are already demanding information regarding their “body burdens”, or the number and amount of chemicals present in their system, as evidenced by the “Human Toxome Project” In conclusion, the EWAS is a promising way to search and consider potential environmental factors as associated with disease or other clinical phenotypes. These results demand a rethinking and restructuring of studies that study disease in the genomics context. The time is ripe to usher in “enviromics” File S1The tables in this file describe the baseline demographics of the NHANES cohorts  per T2D status (Fasting Plasma Glucose >125 mg/dL). T2D cases were determined by a clinical threshold of ≥126 mg/dL fasting blood glucose. Unweighted total samples were similar across cohorts. Age and BMI were significantly different between each of groups and the proportion of sex was significantly different in 3 of the 4 cohorts . Low, medium, and high estimates of SES were computed by tertile of poverty index. Low SES (lowest tertile of poverty index) is also associated with T2D status in 3 of the 4 cohorts (3rd tertile SES referent group). Ethnicity was not seen to be associated with T2D status (“white” ethnicity referent group). * denotes unweighted number. All other statistics are weighted.(0.09 MB DOC)Click here for additional data file.Figure S1Trans-β-carotene vs. Diabetes Status for 2001–2002, 2003–2004, and 2005–2006 cohorts. Raw exposure data  versus T2D Status (Fasting Plasma Glucose >125 mg/dL) for validated environmental factors. Horizontal line represents the weighted median of the group. Cohort plot symbols consistent with (1.17 MB DOC)Click here for additional data file.Figure S2Cis-β-carotene vs. Diabetes Status for 2001–2002, 2003–2004, 2005–2006 cohorts. Raw exposure data  versus T2D Status (Fasting Plasma Glucose >125 mg/dL) for validated environmental factors. Horizontal line represents the weighted median of the group. Cohort plot symbols consistent with (1.01 MB DOC)Click here for additional data file.Figure S3γ-tocopherol vs. Diabetes Status for 1999–2000 and 2003–2004 cohorts. Raw exposure data  versus T2D Status (Fasting Plasma Glucose >125 mg/dL) for validated environmental factors. Horizontal line represents the weighted median of the group. Cohort plot symbols consistent with (0.83 MB DOC)Click here for additional data file.Figure S4Heptachlor Epoxide vs. Diabetes Status for 1999–2000 and 2003–2004 cohorts. Raw exposure data  versus T2D Status (Fasting Plasma Glucose >125 mg/dL) for validated environmental factors. Horizontal line represents the weighted median of the group. Cohort plot symbols consistent with (0.49 MB DOC)Click here for additional data file.Figure S5PCB170 vs. Diabetes Status for 1999–2000 and 2003–2004 cohorts. Raw exposure data  versus T2D Status (Fasting Plasma Glucose >125 mg/dL) for validated environmental factors. Horizontal line represents the weighted median of the group. Cohort plot symbols consistent with (0.50 MB DOC)Click here for additional data file.Table S1Percent of significant  environmental factors found by environmental class and cohort in first stage of T2D association. Number found of total per class is also shown in parentheses. * denotes that there were no factors measured for that particular environmental class as of this writing. There were a total of 21 environmental classes explored.(0.05 MB DOC)Click here for additional data file.Table S2Adjusted odds ratios for validated factors for individuals at risk for T2D diagnosis. Individuals who answered yes to having T2D in the NHANES questionnaire (“Doctor told you have diabetes?”) were omitted from the sample, leaving only those who were at risk for T2D diagnosis. Estimates were adjusted for age, sex, BMI, SES, ethnicity, and cohort. Odds ratios are for a change in 1SD of the logarithm of exposure in association with T2D diagnosis risk.(0.03 MB DOC)Click here for additional data file.Table S3Adjusted odds ratios for validated factors, adjusting for age, sex, BMI, SES, ethnicity, cohort, log10, log10. Odds ratios are for a change in 1SD of the logarithm of exposure in association with T2D.(0.03 MB DOC)Click here for additional data file.Table S4Adjusted odds ratios for validated factors, adjusting for age, sex, BMI, SES, ethnicity, cohort, log10(triglycerides), log10, log10, log10, log10, supplement use (yes/no) . Odds ratios are for a change in 1 SD of the logarithm of exposure in association with T2D. * denotes models did not include β-carotene as these data were not available for the 1999–2000 cohort.(0.03 MB DOC)Click here for additional data file.


The Foot Posture Index (FPI) is a validated method for quantifying standing foot posture, and is being used in a variety of clinical settings. There have however, been no normative data available to date for comparison and reference. This study aimed to establish normative FPI reference values.Studies reporting FPI data were identified by searching online databases. Nine authors contributed anonymised versions of their original datasets comprising 1648 individual observations. The datasets included information relating to centre, age, gender, pathology (if relevant), FPI scores and body mass index (BMI) where available. FPI total scores were transformed to interval logit scores as per the Rasch model and normal ranges were defined. Comparisons between groups employed t-tests or ANOVA models as appropriate and data were explored descriptively and graphically.p < 0.001). There was no difference between the FPI scores of males and females . No relationship was found between the FPI and BMI. Systematic differences from the adult normals were confirmed in patients with neurogenic and idiopathic cavus , indicating some sensitivity of the instrument to detect a posturally pathological population.The main analysis based on a normal healthy population (n = 619) confirmed that a slightly pronated foot posture is the normal position at rest (mean back transformed FPI raw score = +4). A 'U' shaped relationship existed for age, with minors and older adults exhibiting significantly higher FPI scores than the general adult population (F = 51.07, A set of population norms for children, adults and older people have been derived from a large sample. Foot posture is related to age and the presence of pathology, but not influenced by gender or BMI. The normative values identified may assist in classifying foot type for the purpose of research and clinical decision making. Variations in foot posture are thought to influence the function of the lower limb and may therefore play a role in predisposition to overuse injury -4. DespiRecently, a six-item criterion reference tool  was developed in response to a requirement for a quick, easy and reliable method for measuring foot position in a variety of clinical settings . The FPIThe concurrent validity of the FPI has been investigated fully and reported previously . A more The FPI has been used in a variety of clinical and research settings. The applications of the FPI include studies of biomechanical risk factors for neuropathic ulceration in diabetes , identifOne of the limitations of the FPI is that, to date, there have been no normative data available for comparison and reference. The aim of this study therefore, was to establish normative FPI reference values for use in research and to assist in clinical decision making.A search was carried out using online databases  and internet search engines for studies relating to the use of the FPI. The authors of the studies referencing either the eight or six item FPI were contacted via email with a view to capturing the original data. Original, anonymised datasets were received from nine authors in various formats. Observations from 1648 individual participants were provided, originating from 16 studies undertaken in nine centres. Data collated included centre, age, gender, pathology (where relevant), individual item scores for both the left and right foot (where available), FPI (six-item) total scores for the left and right foot , and body mass index (BMI) values, where available.All data provided for the normative analysis was anonymous and local ethical approval had been given for each of the original studies from the relevant institutional research ethics committees. A summary of the datasets obtained is provided in Table t-test for unpaired data or a one-way ANOVA with Tukey's post hoc test as appropriate to the number of factors. Relationships were explored using scatter plots and Pearson's correlation coefficient.The FPI has undergone testing against the Rasch model to determine its internal construct validity . OrdinalReference ranges were defined using the cut points employed previously for similar studies , namely:(i) Normal: values lying in the range, mean +/- 1 standard deviation (SD)(ii) Potentially abnormal: values 1 to 2 SDs from the mean(iii) Pathological: values lying outside 2 SDs from the meanThe total sample comprised 1,648 participants. There were 717 males, 825 females and 116 participants for whom gender was not specified. The mean age was 42.3 years (SD = 25.1) with a range of 3 to 96 years. BMI data were available for 1,101 participants. 1007 of the participants were normals from the control arms of studies, with the remaining 641 having defined pathologies. Only data from normal adults were included in the main analysis, and the data from the pathological groups is reported separately.t-test to identify any side-related systematic difference between observations . This difference was not significant . The mean of the logit scores for the normal sample was 2.4 (SD = 2.3). Logit scores were back transformed into FPI raw scores and normal, potentially abnormal and truly pathological ranges defined. These are presented in Table The normal adult sample comprised 619 observations of a single limb from each participant. Data were first tested for normality and this was confirmed both graphically and by the calculation of skewness and kurtosis statistics . Left and right side data were compared using Student's 1.9 SD = .1, rightt-test for unpaired data. There was no statistically significant difference between the FPI scores of males and females .Data for male and female participants were explored using descriptive statistics and Student's Normative data were explored for age-related trends and initial scatter plotting suggested that FPI scores may vary with extremes of age . The differences between groups were all confirmed as significant by Tukey's post hoc test (p < 0.001). Separate reference ranges have therefore been defined for the minor and older adult groups .The dataset was explored for any evidence of a relationship between the FPI and BMI. Scatter-plotting and calculation of Pearson's correlation coefficient identified no relationship between these two variables  those with miscellaneous local musculoskeletal symptoms n = 399); (ii) a group with diabetic neuropathy (n = 36); (iii) a group with neurogenic pes cavus associated with peripheral neuropathy (n = 44), and; (iv) a group with idiopathic pes cavus (n = 162). The means and standard deviations for the miscellaneous musculoskeletal symptoms group and the diabetic neuropathic group were comparable with the normal population, as would be expected for conditions not normally associated with significant structural change. Conversely, the neurogenic cavus and the idiopathic cavus groups were confirmed as representing a clearly pathological population  The FPI is only one of a number of measures of foot posture currently available. Razeghi and Batt  discuss The FPI has now been employed in several studies and median FPI raw scores for normal samples have been reported to lie consistently around +5 ,25. OtheStatistically determined reference ranges for postural variations such as standing foot position are inherently wide, so must be used as a general guide only in interpreting FPI scores in a clinical context. It is recognised that clinically, relatively minor variations from the mean may increase risk of mechanically induced pathology, although the strength of these relationships have not been confirmed scientifically and certainly vary for different pathological groups. Except for foot postures falling clearly outside the normal range, the reference ranges alone are probably not adequate for clinical decision making.There was some evidence of age-related variation in mean foot posture scores and this is in agreement with previous studies. In the recent study by Scott et al , a samplWhile age was found to have an effect on foot posture there was no evidence of any systematic difference between the FPI scores of males  and females . This is again in agreement with the longitudinal arch study by Staheli et al  who founThe current study found no relationship between BMI and the FPI. Previous studies undertaken using measures such as the footprint angle (FA) and the Chippaux-Smirak index (CSI) have reported lowered longitudinal arches, a broader midfoot area and subsequently flatter feet in people with high BMI values . HoweverIt is known from empirical observation and previous studies that foot posture differences may be encountered in association with underlying disease processes or functional pathology. Comparison of the FPI scores from the normal sample with data from participants known to have identified pathology revealed variations consistent with those predicted by theory. The group with neurogenic pes cavus  and idiopathic pes cavus  had FPI scores significantly different from the normal population (mean logit score = +2.4) indicating that the FPI data was sensitive to disease-related postural changes. Data have also been reported elsewhere indicating the sensitivity of the FPI to postural change associated with pathological pes planovalgus (median FPI raw score = +12) . ConversThere are several limitations to this study that warrant discussion. The most compelling of these is that the data used did not come from a prospectively constructed random sample, such as a general practice or telephone directory derived random sampling frame. Such sampling methods are extremely resource intensive and financially costly whereas the retrospective compilation of a large sample from existing sources covering both normal and pathological subgroups was felt to be a realistic compromise between impact and resource. The dataset was compiled using data from nine centres which raises the possibility of some inconsistency in data collection. One centre had recorded age as a range rather than an integer in years, although sufficient detail was provided to allow classification according to the cut points provided in the analysis. Body mass index was also of less importance to some studies and was not recorded by all centres. However, all incomplete datasets were missing only variables informing the secondary analysis, and for variables of primary importance such as presence or absence of pathology, the dataset was complete.Observations were derived from either FPI-6 total scores, or through the extraction of the six relevant items from studies using the older eight item version of the FPI. All observers were trained using the official FPI user manual, but it is acknowledged that minor variations in interpretation may have occurred and could not have been controlled for. Conversely, the use of data from multiple centres limits the potential for bias in the total sample and could be considered to enhance the validity of the results.In summary, this study has provided a set of normative values for FPI scores in a healthy adult population. The data also provides mean and standard deviation values to act as comparators for future studies in a range of potentially pathological groups. Future studies defining FPI ranges of normal and abnormal explicitly according to resulting pathology would supplement this statistical definition and would be helpful to our understanding to the link between foot posture and mechanical 'overuse' type symptoms. The FPI scores did not vary systematically with gender, side of observation or BMI, although did vary at the extremes of age. FPI scores in groups with confirmed structural pathology were systematically different from normal, indicating some sensitivity of the instrument. This now requires further confirmation in specific pathological groups. Further investment in studies to determine definitive reference ranges for children and older adults may help to complete the picture.Normative data for the FPI obtained from 619 healthy adults has been presented and compared with grouped data from 1,029 further observations. Based on the analyses presented here, it is concluded that foot posture is influenced by age and presence of pathology, but is not influenced by sex or BMI. The use of age-specific reference ranges provided in this paper will assist in classifying foot type for the purpose of research and clinical decision-making.Journal of Foot and Ankle Research. It is journal policy that editors are removed from the peer review and editorial decision making processes for papers they have coauthored.HBM is Editor-in-Chief of the ACR developed the Foot Posture Index, and with HBM, designed the study. YZC coordinated the data capture and statistical analyses. All authors helped draft the manuscript and read and approved the final manuscript.


No overall increased risk of lymphoma associated with antibodies to human herpesvirus-8 was found in 526 lymphomas and 599 controls (odds ratio (OR)=1.04, 95% confidence interval (CI)=0.62–1.75); significant increases were noted for 19 lymphoplasmacytic lymphomas  and nine low-grade lymphoma/lymphoma B-cell NOS . Kaposi's sarcoma (KS)-associated herpesvirus, also known as human herpesvirus-8 (HHV-8), has been shown to be causally associated with KS, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD)  and Madrid. Cases were consecutive patients newly diagnosed with a lymphoid malignancy between 1998 and 2002 and categorised according to the WHO Classification for Neoplastic Diseases of the Lymphoid Tissues  were included in the study, 28 refused to participate, 25 died before the interview, 116 did not provide a blood sample and five cases had no interview. Of 655 eligible controls, 599 (91.6%) were included in the study, 23 refused to participate and 33 did not provide a blood sample. Further details of the study have been described elsewhere (Antibodies against the lytic antigent K8.1 were tested using a enzyme-linked immunoassay (ELISA) as described previously . All positive subjects were confirmed with Western blot.Peripheral blood mononuclear cells (PBMC) were tested for HHV-8 DNA by quantitative PCR in all subjects considered to be seropositive for either anti-K8.1 or anti-LANA as described previously  and 95% confidence interval (95% CI) in order to measure the association between specific variables and the risk of lymphoma. Questionnaire variables were explored for their association with HHV-8 or with case–control status at The study population consisted of 526 lymphoma cases and 599 controls. The average age at entry was 59.7 years among cases and 58.0 years among controls.No differences were observed in the distribution of cases and controls in relation to age, sex, recruitment area, educational level and history of blood transfusion . These patients are excluded in the following results if not otherwise specified.In all, 32 controls (5.4%) and 29 cases (5.8%) were HHV-8 positive (6 cells=305 882), who was coinfected with HIV and one subject HIV negative with a B-cell lymphoma NOS, who had been previously diagnosed with MCD (copy number per 106 cells=24 444).HHV-8 DNA was identified in nine of 69 (13.0%) HHV-8 seropositive subjects, including those HIV-infected subjects and organ recipients and in none of the 132 seronegative subjects matched by age and sex to positive subjects and randomly selected from the pool of negatives. Two subjects showed a high HHV-8 copy number, one patient with a T-cell lymphoma (copy number per 10In our study, no overall differences in the HHV-8 prevalence could be found between cases and controls. However, HHV-8 was strongly associated with two subgroups, a four-fold increased risk of lymphoplasmacytic lymphoma and a five-fold increased risk of low-grade B-cell lymphoma and B-cell lymphoma NOS. The detection of HHV-8 DNA in PBMC showed that only one in seven seropositive subjects had viral DNA detectable in blood. A possible aetiologic association with HHV-8 was suspected in two subjects with a very high viral DNA copy number in PBMC. One was an HIV-positive subject with a T-cell lymphoma. This observation is in agreement with a recent case report of a PEL of T-cell origin associated with HHV-8 was suggested in an HIV-negative patient (In our data, we also observed a slight increased risk associated with HHV-8 for splenic marginal zone lymphomas. These neoplasms can also harbour plasma cells with cytoplasmatic immunoglobulins and must be considered within the range of differential diagnosis of lymphoplasmacytic lymphoma (HHV-8 is not readily isolated in cell culture. HHV-8 DNA can be detected by PCR in all KS tumour biopsies. However, viral DNA is detectable by nested PCR in the blood in only half of KS patients and 10% of asymptomatically infected subjects because few infected cells are present in peripheral blood (Our case–control study was based on hospitalised patients with high participation rates for both cases and controls. Serology was obtained at the time of study entry. A bias in our estimates could take place if the underlying HHV-8 prevalence of the control population could affect hospitalisation rates due to HHV-8-related diseases. Available data from the Spanish general population, however, indicate that the HHV-8 prevalence ranges between 6 and 8% (Overall, our results suggest that HHV-8 is unlikely to contribute importantly to lymphomagenesis. The association with lymphoplasmacytic lymphoma and with low-grade lymphoma deserves further research.


APEX1, XRCC1, XRCC2, XRCC3, XPD) and damage response  were determined. Associations between telangiectasia and genotypes were assessed among 409 patients, using multivariate logistic regression. A total of 131 patients presented with telangiectasia and 28 patients with fibrosis. Patients with variant TP53 genotypes either for the Arg72Pro or the PIN3 polymorphism were at increased risk of telangiectasia. The odds ratios (OR) were 1.66 : 1.02–2.72) for 72Pro carriers and 1.95 (95% CI: 1.13–3.35) for PIN3 A2 allele carriers compared with non-carriers. The TP53 haplotype containing both variant alleles was associated with almost a two-fold increase in risk  for telangiectasia. Variants in the TP53 gene may therefore modify the risk of late skin toxicity after radiotherapy.Breast-conserving surgery followed by radiotherapy is effective in reducing recurrence; however, telangiectasia and fibrosis can occur as late skin side effects. As radiotherapy acts through producing DNA damage, we investigated whether genetic variation in DNA repair and damage response confers increased susceptibility to develop late normal skin complications. Breast cancer patients who received radiotherapy after breast-conserving surgery were examined for late complications of radiotherapy after a median follow-up time of 51 months. Polymorphisms in genes involved in DNA repair ( Radiotherapy is commonly applied after breast-conserving surgery to reduce the risk of locoregional recurrence of breast cancer and has been shown to be as effective as radical mastectomy . The proAs radiation therapy exerts its cytotoxic effects through damage to cells, proteins and DNA, the individual capacity to repair damaged DNA may modify the response of the normal tissue. Radiation-induced DNA damage is diverse and therefore nearly all DNA repair pathways might be involved in its removal, especially repair of double-strand breaks through mechanisms such as homologous recombination and non-homologous end joining . In addip21 is a critical cell-cycle checkpoint gene, regulated tightly by p53. As soon as DNA is damaged by radiation, binding of p53 protein induces transcription of the downstream gene p21, which stops cells from entering into the S phase  were abstracted from the irradiation protocols. As described earlier , in this patient cohort (The late side effects were classified according to the RTOG/EORTC late radiation morbidity scoring schema  supplemeXRCC1 -77 polymorphism (rs3213245) were the PCR primers (sense) 5′-ctttagccagcgcaggtcg-3′OH and (antisense) 5′-ccccatgcaggtccctcac-3′OH, sensor 5′-cccgccccctcccactc-3′-FL and anchor 5′-LC Red640-ccctgcccctcggaccccatactc-3′P. The sense primer included a mismatch to avoid stem loops in the amplicon because of the high and repetitive G/C content of the target sequence. PCR primers and probes were designed with the help of Tib Molbiol . Annealing temperature of the primers was 60°C. The PCR was performed for all polymorphisms with Qiagen reagents  in a volume of 10 μl using 10 ng of DNA. Overall, 10% randomly selected samples were analysed by conventional PCR-RFLP to verify the LightCycler results; 100% concordance was found. The insertion of the TP53 PIN3 polymorphism was identified by standard PCR and electrophoresis (Most polymorphisms (see Table 2) were detected by amplification with real-time PCR followed by melting-curve analysis with fluorescence-labeled hybridisation probes in a LightCycler  as described earlier  and 95% confidence intervals (CI) were computed using the LOGISTIC procedure in SAS version 9.1 . Possible effect modification of genotype associations by other covariables was evaluated by the log likelihood ratio test comparing models with and without the first-order interaction terms. All tests were two-sided and considered to be statistically significant with a P-value of ⩽0.05. The logistic regression analysis was performed only for the occurrence of telangiectasia, excluding seven patients who developed fibrosis with a score ⩾2 but not telangiectasia. Multivariate models included NTD, age at the time of late toxicity evaluation, and follow-up time since end of radiotherapy. Stepwise backward elimination with P⩽0.03 as threshold was used to develop a final model to control for potential confounders. The final model included, in addition hospital facility, acute skin toxicity, a history of hypertension and allergy, skin type (three categories), pack-years of smoking , and marital status.Significant differences in distribution of genotypes by presence of late skin toxicities (scores ⩾2) were tested by the P<0.05 although 13 SNPs were tested.Analyses to assess association between haplotypes and risk for telangiectasia were carried out using the function haplo.glm of the R package haplo.stats, which uses a generalised linear model (glm) allowing for an ambiguous linkage phase . Carriers of the TP53 PIN3 A2 allele were also at increased risk of telangiectasia . None of the other genetic polymorphisms studied showed significant associations with occurrence of telangiectasia.We found a significant association between genetic polymorphisms in the iectasia . CompareTP53 Arg72Pro and TP53 PIN3 polymorphisms (P<0.001). We therefore investigated haplotype effects of the two TP53 polymorphisms. Compared with the common ArgA1 haplotype, the ProA2 haplotype containing both variant alleles was associated with a significantly increased OR of 1.97 (95% CI: 1.11–3.52) for telangiectasia  was found between the iectasia . HaplotyTP53 on risk for telangiectasia, according to occurrence of acute skin toxicity (moist desquamation). Thirty women (22.9%) had presented with acute skin toxicity during radiotherapy in patients with telangiectasia, and 45 women (16.2%) in those without telangiectasia. The elevated risk of telangiectasia associated with the TP53 ProA2 haplotype was found only in patients who did not present with acute toxicity during radiotherapy  and not in those who experienced acute skin toxicity during radiotherapy (Pheterogeneity=0.06)  .TP53 were associated with an increased risk for developing telangiectasia after radiation therapy. Although both variants, TP53 72Pro and PIN3 A2, were associated with elevated risk, the haplotype results suggested that cis effects of the two variants may be most relevant.In this study of breast cancer patients treated with radiotherapy after breast-conserving surgery, we found that variants of TP53 variants in codon 72 showed that this polymorphism might modulate these two responses. The 72Arg form induced apoptosis more efficiently than the 72Pro form. In contrast, the 72Pro form appeared to induce a higher level of G1 arrest than the 72Arg form giving time to repair (Two of the many p53 functions may be important in modulating radiosensitivity. Growth arrest mediated by p53 plays an important role in inhibiting mitotic cell death in epithelia of the small intestine of mice and, thus, is thought to reduce radiation toxicity in these animals (o repair . Consequo repair . AlthougTP53 PIN3 polymorphism on the risk of late radiation toxicity, but the results of the haplotype analysis suggested the strongest effect on risk conferred by the haplotype containing both variant alleles. The functional significance of TP53 PIN3 has remained largely unexplored. Our haplotype analysis revealed further that the strongest risk effect of the 72ProA2 haplotype was visible in patients who did not develop severe acute side effects during radiotherapy. We proposed that the Pro allele carriers experienced reduced cell loss by apoptosis and, potentially, mitotic catastrophe during therapy and were, thus, protected from severe acute side effects as we found in our analysis of acute side effects (in vivo and in vitro effects of the TP53 Arg72Pro and PIN3 polymorphisms are needed, however, before we can apply these TP53 variants as predictive markers for late side effects of radiotherapy.We also observed an independent effect of the p21 Ser31Arg polymorphism on the risk of late skin toxicity. Other studies also failed to find an association of this variant with risk or prognosis of breast cancer (p21 plays a direct role in mediating irradiation-induced G1 arrest, with p53 as the transcription factor in this process. This mechanism indicates a possible combined effect of polymorphisms in the two genes. However, p53 may modulate response to radiation damage in the G1 phase of the cell cycle through mechanisms independent of p53-mediated transcriptional activation of p21 and cell-cycle arrest (XRCC1 Arg399Gln polymorphism has been reported to be associated with telangiectasia but not with fibrosis, particularly in patients who did not receive a boost, albeit based on 167 patients of whom 39 presented with telangiectasia (XRCC1 genetic polymorphisms, -77T>C, Arg194Trp, Arg280His, and Arg399Gln (XRCC3 and APEX1 were studied in breast cancer patients receiving radiotherapy (summarised in In addition, ten polymorphisms causing an amino acid change in six different DNA repair genes were investigated for associations with telangiectasia, but no significant effects were detected. The TP53 genetic variants as predictors of late tissue reactions to radiation therapy. However, both the TP53 codon 72 and intron 3 variants have been found to be associated with poorer prognosis of non-small cell lung cancer (To our knowledge, this is the first epidemiological study on the two This study has a number of strengths. Breast cancer patients from this cohort were treated similarly, with radiation dosage carefully assessed, and patients were followed prospectively. Improved radiation techniques at the time of patient recruitment, as well as retrieval of individual irradiation dose methods and records, allowed for proper calculations of BED. The phenotype was precisely defined using the standardised scoring system for late toxicity. In addition, we accounted for patient- and treatment-related factors that influenced risk for telangiectasia when assessing the effect of the genetic variants.Both telangiectasia and subcutaneous fibrosis are among the most common long-term skin side effects of radiation therapy. Owing to differences in physiological response to radiation of the various skin layers involved and thereby possible differing genetic susceptibility, we opted to restrict the present analysis to telangiectasia because of the limited occurrence of fibrosis and therefore restricted power. Progressive nature of these complications, together with longer time to follow-up, may permit later analyses of late normal tissue complications in this cohort in the future.TP53 gene are associated with risk of late skin toxicity after accounting for patient-related factors and treatment modalities. As this is the first report on the involvement of p53 in late skin adverse effects, replication of these findings in other studies is encouraged. Advances in the search for biomarkers of radiation-induced late skin side effects may lead to improved treatment choices for breast cancer patients, and improve cosmetic outcome as well as quality of life after surviving breast cancer.In conclusion, this prospective study showed that variants in the


Attachment theory predicts and subsequent empirical research has amply demonstrated that individual variations in patterns of early attachment behaviour are primarily influenced by differences in sensitive responsiveness of caregivers. However, meta-analyses have shown that parenting behaviour accounts for about one third of the variance in attachment security or disorganisation. The exclusively environmental explanation has been challenged by results demonstrating some, albeit inconclusive, evidence of the effect of infant temperament. In this paper, after reviewing briefly the well-demonstrated familial and wider environmental influences, the evidence is reviewed for genetic and gene-environment interaction effects on developing early attachment relationships. Studies investigating the interaction of genes of monoamine neurotransmission with parenting environment in the course of early relationship development suggest that children's differential susceptibility to the rearing environment depends partly on genetic differences. In addition to the overview of environmental and genetic contributions to infant attachment, and especially to disorganised attachment relevant to mental health issues, the few existing studies of gene-attachment interaction effects on development of childhood behavioural problems are also reviewed. A short account of the most important methodological problems to be overcome in molecular genetic studies of psychological and psychiatric phenotypes is also given. Finally, animal research focusing on brain-structural aspects related to early care and the new, conceptually important direction of studying environmental programming of early development through epigenetic modification of gene functioning is examined in brief. Early attachment, a specific personal relationship developing between an infant and the caregiver has been considered essential for survival as well as for later physical and mental development in primates including the human species . The humAttachment relationships are formed in the course of interactions with caregivers. Infants accumulate information regarding readiness, quality and reliability of responses from others and, by the end of the first year of life, specific representations are formed about the caregivers, the self and the nature of relationships. These representations are theorised to influence children's behaviour contemporaneously, as well as influencing their subsequent social relationships .Secure (B) attachment can be characterized by the infants' open communication of emotions and their ability to make use of the caregiver as a secure base from which to explore. There are two organized insecure attachment patterns; infants avoiding contact or interaction with the caregiver upon reunion and minimizing the expression of negative emotions under stress are termed avoidantly attached (A), whilst infants expressing intense negative emotions and wanting contact, but unable to settle following the separations are classified as resistantly attached (C). Infants who are unable to obtain comfort from the caregiver at times of fearful arousal, and whose behaviour in the reunion episodes appears to lack a clear strategy, may display simultaneous or sequential contradictory behaviours, misdirected, stereotypical movements, extended freezing and direct expression of fear in the presence of the caregiver. These behaviours are rated on a 9-point scale and those with a summary score > 5 are classified as disorganized (D).Individual variations in infants' attachment to caregivers can be observed through their behaviour, especially under conditions of stress that activate seeking and maintaining proximity. The Strange Situation Procedure (SSP)  developeThousands of assessments during the last three decades have allowed the estimation of the frequencies of these infant attachment types across a range of populations. Frequencies in low social risk community samples are typically ~55% secure, ~15% avoidant, ~10% resistant and ~15% disorganized. There is some cross-cultural variation in these frequencies, but differences within any culture can be just as large as between cultures . In highcaregivers' behaviour. In their seminal study [Bowlby's attachment theory is a truly environmental theory as it has explained individual differences in attachment patterns (attachment types) by individual variations in al study , AinsworAlthough many studies demonstrated a significant link between early care and attachment, studies varied greatly regarding in estimates of the strength of the relationship. De Wolff and van IJzendoorn  revieweddemographic risk factors, especially if accumulated may effect the development of attachment, presumably through their proximal or distal influence on parenting [Over the last three decades, it has been shown that different arenting . Income arenting -19.psychological health of parents, especially mothers. Empirical studies have provided contradictory results, but the majority found that mothers depressed postnatally were more likely to develop insecure attachment relationships with their infants [social support systems [mental state with respect to close (attachment) relationships. In several studies, the security of maternal attachment representations, as assessed by the Adult Attachment Interview (AAI) [It has been expected that secure attachment is promoted by the  infants -23. Stud systems , but in  systems -26, whil systems ,28. Secuew (AAI) , has beeew (AAI) .It has also been shown that while isolated individual risk factors may not have a significant effect on parent-child attachment, the accumulation of adversity may result in sub-optimal relationship development and insecurity of infant attachment . Raikes can be an environmental risk factor for attachment security and compromised later development.Friedman and Boyle  have recAttachment disorganisation became a focus of developmental research when rarely occurring incoherent and contradictory infant behaviours, not fitting the Ainsworth categories, appeared to be predominant among maltreated or otherwise deprived groups of infants and young children ,40. InfaNewborns' biologically based capabilities of self-regulation of arousal and distress states have an immediate impact on parents. The great individual variation in these capabilities can be described by dimensions such as the infant's disposition for distress or negative emotionality, irritability and soothability ,58. TherSeveral studies have found that newborn behavioural measures are related to later secure and insecure attachment classifications -70. ThesSince the study of parent-child attachment was so strongly driven by the environmental theory, a quantitative behaviour-genetic approach has only recently been used to investigate heritable and environmental variance components of attachment security. Most of the existing twin studies of attachment overviewed here have not been designed to focus on attachment, therefore they are quite heterogeneous regarding children's age and the method of assessment. In the Louisville Twin Study (LTS), Finkel and Matheny  found a It is important to recognize the power constraints of quantitative genetic studies such as those using twin comparisons. These analyses aim at assessing heritability; that is, estimating how much of the population variance is due to genetic effects . The twin method is based on comparison of twin correlations or concordances. If genetic effects are important for the trait in question, then correlation or concordance between monozygotic twins should be significantly greater than that between dizygotic twins. Limited sensitivity is inherent in the methodology based on detecting significant differences between twin correlations. This is not such a great limitation for many psychological and psychiatric phenotypes with substantial heritabilities of around 0.5, but may cause problems in detecting smaller, yet potentially important genetic effects. (It is estimated that any specific gene effect accounts for less than 1% of the variance of complex traits .) The poIn addition, behaviour-genetic analyses usually employ main effects models dividing up the total phenotypic variance into additive (or dominant) genetic, shared and non-shared environmental components. They normally do not separate gene-environment interactions (genetic sensitivity to environments) and gene-environment correlations (arising from experiences correlated with genetic propensities). Finally, quantitative genetic modelling does not go beyond the extent to which genetic factors influence behavioural traits, that is they are not informative about specific genes or specific environmental factors affecting the behaviours in question.The development of molecular genetic methodology started to shift the focus of study towards locating and identifying specific genes underlying genetic effects evident in twin and adoptive studies, even before sequencing the human genome was completed. The widely used strategy of allele association investigates correlations of gene variants with phenotypes, i.e., it probes if individuals with a trait of interest carry a specific gene variant more frequently than individuals in a control group (case-control studies). The method of allele association has been quickly and productively applied in the area of multi-factorial mental illnesses for which genetic components have long been demonstrated by large heritability estimates. Genes regulating the cerebral levels of important neurotransmitters  or signal transmission efficiency (neurotransmitter receptors and genes) have been targeted in association studies of major psychiatric disorders such as schizophrenia, bipolar disorders, attention deficit/hyperactivity disorder (ADHD), and autism , as wellThe first investigation of the specific genetic background of attachment behaviour showed an association between D4 dopamine receptor (DRD4) gene polymorphism and infants' attachment behaviour . The levIn the longitudinal Budapest Infant-Parent Study (BIPS), a relative over-representation of the 7-repeat variant of the 48 bp VNTR polymorphism was found in the group of infants displaying insecure-disorganised attachment behaviour with their mothers in the Strange Situation . The estSpecific genetic effects on phenotypes may be conditional on specific environments and thus be undetected in other environments. Similarly, exposure to specific environments are often influenced by choices that depend on the individuals' genetic make-up. Such interplay between genes and environment may channel individual development into different trajectories early in life and affect the long-term development of mental health and disorder. A shift from association studies targeting genetic main effects towards investigating the interaction of genetic and environmental influences is reflected by the accumulating evidence for such processes being reported in recent literature . ImportaOur own study of the genetic effects on disorganised attachment has been extended by investigating the interplay between DRD4 gene polymorphism and maternal behaviour . In ordeResults of this first molecular genetic study of infant attachment ,96 seem Studies have been extended by investigating associations with other candidate genes. The polymorphic serotonin transporter gene, coding for one of the important regulator of the synaptic level of the neurotransmitter serotonin, has been linked previously to anxiety-related traits  and affeThe gene-environment interaction effects on attachment reported in the above-cited publications are consistent with Belsky's differential susceptibility hypothesis , i.e., cFollowing up their above mentioned study , KochansIn a small-scale study, (N = 47), infants' DRD4 genotype was found to moderate the relationship between maternal insensitivity and externalising child behaviour: children who carried the 7-repeat allele and had relatively insensitive mothers showed the highest level of externalising behaviour . In a fuGenetic association studies in the field of psychiatric and psychological genetics have been suffering from inconsistencies in replications of results. There can be many reasons for these difficulties, some of which are the result of methodological flaws, for example genotyping errors. Improved measurement of phenotypes and carefully controlled genotype determination can reduce the danger of false negative findings . FailureAs mentioned above, some of these problems can be tackled by increasing study sizes and combining study samples, increasing the number of carefully chosen candidate polymorphisms or as Plomin and Davis suggest  employinStudies in animal models have found convincing evidence for the critical impact of early emotional experiences. Studies from the 1950s showed that even short separations of young rodent pups from their mother have profound and persistent effects on behaviour and physiological stress reactivity . In the An avian model of early parent-offspring bonding is filial imprinting in precocious birds which is accompanied by extensive reorganization in the frontal lobe. Domestic chicks imprinted on artificial stimuli in experimental settings showed increased synaptic connectivity in the intermediate medial hyperstriatum ventrale (IMHV), which seems to be important for storage of memory acquired during imprinting ,126. BocThis experience-dependent development of neural connections serves the adaptive response of the offspring to its actual normative environment. However, in case of adverse early influences, the same plasticity may lead to altered neural development with long-lasting behavioural and physiological effects. There has been accumulating evidence for the importance of parental care, especially tactile stimulation in the subsequent development of infants of mammalian species. Separation from the mother in rodents induces physiological and behavioural responses including vocalisation and searching behaviour, corticosterone hormone release and inhibition of metabolism related to growth and later stress reactivity ,130. BocThese few examples selected from an extensive research field illustrates the critical effects of early stress on brain development. The mechanisms underlying alterations in brain development are far from being understood in animals and even less in humans, but new molecular techniques and imaging methods may hold the key to growing knowledge of normal and corrupted developmental trajectories.without a change in the inherited nucleotide sequence. Epigenetic modifications of promoter regions that influence the transcription of genes coding for crucial protein products can be induced by the environment. Although the mechanisms through which these modifications occur have yet to be fully explored, one important process is environmentally induced DNA methylation leading to transcriptional silencing. It has also been shown that these environmentally mediated effects on gene expression tend to persist in the individual's lifetime and can even be transmitted from one generation to the next [In relation to the association of infant attachment with a promoter polymorphism of the serotonin transporter gene, we have mentioned that gene expression may be affected by variation in the DNA sequence of the regulatory region of the gene. There is, however, increasing evidence for changes in gene expression through DNA and chromatin modifications the next . The piothe next  has showthe next . Moreovethe next ,138. Resthe next . Thus, tThere is a considerable individual variation in infants' attachment behaviour with their primary caregivers. Attachment theory, as first conceived, gave an environmental explanation for this variation, regarding caregivers' sensitive responsiveness to infant signals as the crucial factor for the development of optimal, secure attachment. This exclusively environmental explanation has recently been challenged by studies that have included physiological and genetic measures. Although twin studies of attachment are providing mixed results, molecular genetic studies are showing that specific genetic polymorphisms of the dopaminergic and serotonergic neurotransmisson systems may moderate the statistically identified links between caregivers' behaviour and infant attachment. These gene-environment interaction effects, pointing to genetically-based differential susceptibilities to care-giving environments, have been more often found for disorganised attachment, which is an extreme form of insecure attachment predictive of subsequent behavioural problems and psychopathology. Because the results of the as-yet few and relatively small-scale studies are not wholly consistent, there is a clear need for larger-scale and carefully designed studies that examine multiple polymorphisms of multiple candidate genes. More recent research on the epigenetic modification of gene expression by early maternal care in animals suggests the possibility of similar processes affecting human development. The investigation of such processes in humans, although not feasible at present, would offer an opportunity to gain a deeper understanding of developmental psychopathology and the intergenerational transmission of attachment and parenting.The author declares that they have no competing interests.


APOC3 gene have been associated with hypertriglyceridemia and may impact on phenotypic expression of the metabolic syndrome (MetS). The rs7566605 marker, located near the INSIG2 gene, has been found to be associated with obesity, making it also a potential genetic determinant for MetS. The objective of this study is to examine the APOC3 -455T>C and the INSIG2 rs7566605 polymorphisms as potential genetic determinants for MetS in a multi-ethnic sample.Common polymorphisms in the promoter of the APOC3 -455T>C and INSIG2 rs7566605 polymorphisms, and classified for the presence or absence of MetS (NCEP ATP III and IDF definitions). The total study population included 2675 subjects (≥18 years of age) from six different geographical ancestries.Subjects were genotyped for both the APOC3 -455C were more likely to have MetS (NCEP ATP III definition) than noncarriers . The basis of the association was related not only to a higher proportion of -455C carriers meeting the triglyceride and high-density lipoprotein cholesterol criteria, but also the blood pressure criteria compared with wild-type homozygotes. Plasma apo C-III concentrations were not associated with APOC3 -455T>C genotype. The INSIG2 rs7566605  polymorphism was not associated with MetS or measures of obesity.For the overall study population, the prevalence of MetS was 22.6% (NCEP ATP III definition). Carriers of ≥1 copy of APOC3 was associated with an approximately 2-fold increased risk of MetS, whereas the INSIG2 rs7566605 polymorphism was not associated with MetS.Meta-analysis of the sample of multiple geographic ancestries indicated that the functional -455T>C promoter polymorphism in  The metabolic syndrome (MetS) is a clinical entity characterized by abdominal obesity, hypertension, hypertriglyceridemia, low plasma high-density lipoprotein (HDL) cholesterol and elevated glucose ,2. MetS APOC3), one of the most studied genes in lipoprotein metabolism. APOC3 encodes a 79-amino-acid glycoprotein produced mainly in the liver, which acts as a constituent of triglyceride-rich lipoprotein particles, inhibiting the action of lipoprotein lipase and interfering with receptor-mediated lipoprotein uptake. The common -455T>C single nucleotide polymorphism (SNP) of the APOC3 gene, located within an insulin-response element in the promoter region, has previously been found to be associated with elevated plasma triglyceride concentrations in multiple populations ). Furthermore, upon repeating the meta-analysis with the IDF definition of MetS, a modest but significant decreased risk was found overall for INSIG2 rs7566605 C allele carriers , contrary to the expected direction of risk. These negative results again are a cautionary reminder of the great risk of false positive results in genome-wide association studies and stress the importance of replication and the use of functional markers [Our study also examined the rs7566605 genetic variant, located 10 kb upstream of the  obesity . No asso obesity -14 that  obesity . If anyt markers .APOC3. This association might be due to a direct effect of the genetic variation or to linkage disequilibrium with other functional changes. No association, however, was found for the INSIG2 rs7566605 marker with measures of obesity or with MetS. Finding a consistency in association between MetS and APOC3 in a multi-ethnic study group, including populations which differ considerably in MetS prevalence, strengthens the probability that the -455T>C APOC3 promoter polymorphism plays a key role in MetS expression. The findings suggest that factors involved in metabolism of triglyceride-rich lipoproteins may be important in the development of MetS and could represent markers for diagnosis or perhaps sample stratification for interventions.In summary, we report an association between MetS and a common promoter variant in The author(s) declare that they have no competing interests.RLP participated in the experimental design, data acquisition and analysis, interpretation of results, and manuscript writing. MRB participated in the analysis of the data. TKY, PB, SSA, SY, BZ, SBH, AJGH, PWC and MWH were involved in the provision of patient samples and/or clinical data. SSA and MWH participated in the manuscript writing. RAH participated in the experimental design, data analysis and interpretation of results and manuscript writing. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:APOC3 -455T>C polymorphism. Table S3. Genotype and allele frequencies for the APOC3 -455T>C polymorphism in subjects with and without MetS. Table S4. Clinical and biochemical data of subjects when classified in accordance to their genotype of the INSIG2 rs7566605 G>C polymorphism. Table S5. Genotype and allele frequencies for the INSIG2 rs7566605 G>C polymorphism in subjects with and without MetS. Table S6. Greenland Inuit plasma apo C-III concentration, by APOC3 -455T>C genotypeSupplementary Tables. Six supplementary tables including: Table S1. Clinical and biochemical data of subjects. Table S2. Clinical and biochemical data of subjects when classified in accordance to their genotype of the Click here for file


In pregnancy, maternal serum concentrations of calcitriol significantly rise as a result of increased renal and placental contribution in order to assure calcium supply for the developing fetus. Considering that placenta is a site for vitamin D activation, and the versatility and potency of calcitriol, it is feasible that this hormone participates in fetal/placental development and physiology. In the present work we studied calcitriol actions upon human chorionic gonadotropin (hCG) secretion and expression in cultured trophoblasts, as well as vitamin D receptor (VDR) and CYP27B1 immunolocalization in placental villi.Quantification of hCG in culture media was performed by immunoassay. Expression studies were carried out by real time PCR. Analysis of CYP27B1 and VDR localization in placental slides were performed by immunohistochemistry. Statistical significance was established by one way ANOVA using Tukey test for comparisons.Calcitriol regulated hCG in a time-dependent manner: at 6 h the secosteroid stimulated hCG, whereas longer incubations (24 h) showed opposite effects. Interestingly, calcitriol stimulatory effects on hCG were accompanied by an increase in intracellular cAMP content and were abolished by pre-incubation of the cells with a selective protein kinase A inhibitor. Immunohistochemical techniques showed differential VDR localization in the syncytiotrophoblast layer or in the vascular smooth muscle cells depending on the epitope to which the antibodies were raised . CYP27B1 was immunolocalized in the syncytiotrophoblast layer of placental villi.The presence and location of the vitamin D activating enzyme CYP27B1 as well as the specific receptor for vitamin D were shown in placental sections. The latter, together with findings demonstrating specific effects of calcitriol acting through the VDR and the cAMP/PKA signaling pathway upon hCG expression and secretion, indicate that there is a functional vitamin D endocrine system in the placenta, and recognize calcitriol as an autocrine regulator of hCG. Calcitriol, the most active vitamin D metabolite, exerts its biological effects by binding to the vitamin D receptor (VDR), which is a ligand-activated transcription factor that recognizes cognate vitamin D response elements (VDREs) in target genes, and can also elicit rapid responses mediated by membrane receptors , containing 20% heat-inactivated FBS. Incubations were performed in humidified 5% CO2-95% air at 37°C. The morphological aspects of cells were examined daily, secreted hCG was measured by immunoassay (EIA) following manufacturer instructions and results were normalized against total protein content. Protein was determined by the method of Bradford 25-hydroxyvitamin D bioconversion into [3H]1,25-dihydroxyvitamin D was significantly reduced in preeclamptic placentas [The concentration of hCG was also measured after 12, 24 and 48 h of calcitriol treatment, but the results reported in the present study were only those that differed significantly when compared with the vehicle alone. The stimulatory effects observed at 6 h were no further evident after 12 or 24 h, and when cells were incubated in the presence of calcitriol during 2 consecutive days, the effects were rather inhibitory. Inhibition was evident at the mRNA level after 24 hours treatment, preceding the observed response in hCG protein. These data are probably more likely to be reflective of the true biological situation. Indeed, our results that calcitriol inhibited hCG were in line with previous data from this and other laboratories where low serum calcitriol and high serum hCG levels were found in preeclampsia , that colacentas , may suglacentas , this melacentas , which pIn non pregnant women the physiological concentration of calcitriol fluctuates between 40–100 pM. In the present study the calcitriol doses tested were: 100 pM, 1 nM and 10 nM. The lowest concentration (100 pM) is within the physiological range of circulating calcitriol levels in mexican pregnant women (127 pM and 151 pM) as observed previously ,29. The Placenta is considered not only as a source but also as a target of calcitriol . In ordeIn summary, the present study broadens the knowledge of placental vitamin D endocrine system by demonstrating the physiological effects of calcitriol on an important biochemical placental function marker such as hCG. In addition, this is the first report to show immunoreactive VDR in different locations in human placental villi, and opens the field to address important research questions on the role of calcitriol in specific placental structures.The author(s) declare that they have no competing interests.DB carried out real time PCR's analysis, hCG quantification and participated in the design of the study and statistical analysis. EA participated in the design of the study, particularly the molecular studies, performed real time PCR analysis and helped to draft the manuscript. GH performed placenta collection, trophoblast primary cell cultures, RNA extraction and reverse transcription reactions. IM and LG were in charge of all experiments concerning cAMP, including design and analysis of the results. AH contributed in interpretation of data and was involved in drafting the manuscript. FL made substantial contribution to the design of the study, was involved in drafting the manuscript and revised it critically. AM performed the immunohistochemical studies and helped to draft the manuscript. LD conceived the study, participated in the design and coordination, structured the manuscript and actively participated in experimental procedures. All authors read and approved the final manuscript.


AGT gene has been related to an increased risk of hypertension. This finding may also suggest an increased risk of coronary heart disease (CHD).The M235T polymorphism in the P = 0.28). This result was not changed by adjustment  nor by using dominant, recessive and pairwise genetic models. Analyses for AMI risk under the additive genetic model also did not show any statistically significant association . To evaluate the association, a comprehensive systematic review and meta-analysis were undertaken of all studies published up to February 2007 . The meta-analysis (38 studies with 13284 cases and 18722 controls) showed a per-allele odds ratio (OR) of 1.08 . Moderate to large levels of heterogeneity were identified between studies. Hardy-Weinberg equilibrium (HWE) violation and the mean age of cases were statistically significant sources of the observed variation. In a stratum of non-HWE violation studies, there was no effect. An asymmetric funnel plot, the Egger's test (P = 0.066), and the Begg-Mazumdar test (P = 0.074) were all suggestive of the presence of publication bias.A case-cohort study was conducted in 1,732 unrelated middle-age women  from a prospective cohort of 15,236 initially healthy Dutch women. We applied a Cox proportional hazards model to study the association of the polymorphism with acute myocardial infarction (AMI) (n = 71) and CHD. In the case-cohort study, no increased risk for CHD was found under the additive genetic model (hazard ratio [HR] = 1.20; 95% confidence interval [CI], 0.86 to 1.68; AGT gene. However, the relevance of this weakly positive overall association remains uncertain because it may be due to various residual biases, including HWE-violation and publication biases.The pooled OR of the present meta-analysis, including our own data, presented evidence that there is an increase in the risk of CHD conferred by the M235T variant of the  AGT gene, encoding a threonine instead of a methionine at residue 235 of the mature protein, has been associated with a higher plasma AGT level and higher BP in patients homozygous for the T allele and occurs among various ethnic populations Angiotensinogen (AGT) is a liver protein that interacts with renin to produce angiotensin I, the pro-hormone of angiotensin II. Angiotensin II is the major effector molecule of the renin-angiotensin-aldosterone system (RAAS) and plays a key role in the regulation of blood pressure (BP) by increasing vascular tone and promoting sodium retention. Genetic variants in the angiotensinogen gene modify the plasma concentration of angiotensinogen, which has been directly related to arterial blood pressure AGT variant with increased CHD risk; however, this relationship was not confirmed in several other studies AGT gene  with acute myocardial infarction (AMI) and CHD in a large population-based cohort of middle-aged Dutch women and conducted an updated meta-analysis of the available studies to clarify the role of the M235T polymorphism in CHD risk.Given the importance of hypertension in the occurrence of coronary heart disease Study design, general questionnaire, anthropometric and laboratory measurements have been described in detail elsewhere st 2000 were selected as cases. These were 211 CHD cases, including 71 AMIs. For all case subjects, follow-up ended at the date of diagnosis or at the date of death due to cardiovascular disease.We applied the case-cohort design introduced by Prentice Genetic analysis was performed at the Cardiovascular Genotyping (CAGT) laboratory of the Department of Internal Medicine of the University Hospital Maastricht. Genomic DNA was extracted from buffy coats using the QIAamp® Blood Kit . Genotyping of the polymorphisms was performed using a multilocus genotyping assay for candidate markers of cardiovascular disease risk  2 test among the controls. Allele frequencies were estimated by gene counting. We used the ANOVA F test to estimate relationships among the M235T genotypes and continuous variables, while we tested the significance of any difference in proportions by applying the χ2 statistic. A p-value <0.05 (2-sided) was considered statistically significant.Hardy-Weinberg equilibrium (HWE) was tested with the χAGT gene with the outcome, we used a Cox proportional hazards model with an estimation procedure adapted for case-cohort designs. We used the unweighted method by Prentice http://lib.stat.cmu.edu/general/robphreg. A previous meta-analysis AGT M235T variant on its intermediate phenotype (plasma angiotensinogen level) follows an additive model according to the number of T alleles [5% (95% CI: 2 to 8%) increase for the MT and 11% (95% CI: 7 to 15%) increase for the TT genotype versus the MM genotype]. Therefore, our priori hypothesis was that the association between the M235T polymorphism in the AGT gene and CHD follows an additive model according to the number of T alleles. However, other genetic models were evaluated as well. We considered different modes of inheritance as follows: the additive “per-allele” model, the T allele was compared between cases and controls by assigning scores of 0, 1, and 2 to homozygotes for the M allele, heterozygotes, and homozygotes for the T allele, respectively; the recessive model, the TT genotype versus the MT and MM combined genotypes; and the dominant model, the MT and TT genotypes combined versus the MM genotype. We also performed separate pairwise comparisons of the MT and TT genotypes versus the MM genotype.To assess the relationship of the M235T polymorphism in the AGT gene and CHD. Terms used for the search contained both medical subject heading terms and text words: (Met235Thr OR M235T OR T704C) AND (angiotensinogen OR AGT) AND (polymorphism OR mutation OR genetic OR genotype) AND . We also retrieved additional studies by hand searching the bibliographies of original research reports and review articles and through the MEDLINE option “related articles”. Search results were limited to articles published in English and studies on human subjects.We searched PubMed/MEDLINE, Web of Science, and EMBASE up to February 2007 for observational studies evaluating an association between the M235T polymorphism in the AGT M235T genotype frequencies were provided by case-control status (studies without controls were excluded); (ii) risk of CHD or MI was evaluated (studies on recurrent coronary events were excluded); (iii) relevant data were presented to calculate the effect size and its 95% CI; (iv) non-overlapping data were contained. For duplicate publications, the study with the smaller data set was excluded.All studies were considered potentially eligible if they aimed to investigate the relationship between the M235T genotypes and risk of CHD or MI. Any observational study, regardless of sample size, which fulfilled the following criteria, was included: (i) AGT gene genotypes based on different genetic models . We again considered a dominant, a recessive, an additive “per-allele” model and pairwise comparisons. Data were extracted independently and entered into separate databases by two authors (performed by MHZ and MLB). Results were compared, and disagreements were resolved by a consensus.The following information was extracted from each study that we included: the first author's name; country; year of publication; the population evaluated; study design; mean age or age range for case-patients and controls; definition and number of cases and controls; allele frequencies and genotype distribution in case-patients and controls ; consistency of genotype frequencies with HWE ; gender in the evaluated population and male percentage, matching variables, use of blinding of genotyping staff, performing regenotyping of a random sample, and crude ORs and 95% CIs for development of CHD or MI related to the priori hypothesis was the additive model. In each study, we tested for HWE by using the χ2 test or an exact test among the controls by using the genhwi command in Stata 9.2 The method of Mantel-Haenszel was used to calculate the odds ratio for the pooled data in a fixed-effects model, and, if there was evidence for heterogeneity, the DerSimonian-Laird method was used for the pooled odds ratio in a random-effects model, under pairwise comparisons of the different genotypes and dominant, recessive, and additive inheritance models. For all the models used, the T allele was considered the risk allele. The genetic model to be considered as the 2 – based Q statistic for between-study heterogeneity, which is considered to be significant for P<0.10, as well as the 2I statistic for estimation of inconsistency in meta-analyses 2I represents the percentage of the observed between-study variability due to heterogeneity rather than to chance. It ranges between 0% and 100%, where a value of 0% indicates no observed heterogeneity, and larger values indicate an increasing degree of heterogeneity  In addition, we used Cochran's χP<0.10. Meta-analysis was carried out using STATA 9.2. We used random effect meta-regression models with restricted maximum likelihood estimation to evaluate the extent to which different variables explained heterogeneity among the individual ORs. The pre-specified characteristics for assessment of sources of inter-study heterogeneity were: study size  under different genetic models, odds ratios, and variances were corrected by using the HWE-predicted genotype counts in the control instead of the observed counts as previously suggested 2 = 0.020; P = 0.89). General and clinical characteristics of CHD cases and controls are shown in The general characteristics of the randomly sampled participants of the cohort (N = 1522) are given in P = 0.28), which did not alter after adjustment . The same was true for other comparisons . AGT T235M polymorphism with CHD risk under different genetic contrasts. When a recessive model was evaluated, a significant association was found between individuals homozygous for the T allele (T235T genotype) and CHD risk, when compared to carriers of the M allele . Under the dominant model, the association was not significant. Under pairwise comparisons, there was a significant modest association between the T235T genotype and CHD risk, as compared with the M235M genotype . There was evidence for moderate to large between-study heterogeneity under all models . Next, single covariates were added in a series of univariate models. We performed the regression analysis for ten pre-defined potential sources of heterogeneity, including ethnicity, sex, mean age of cases, study size, case definition, source of controls, HWE-violation, blinding in genotyping, performing a sub-sample regenotyping, and matching (we hypothesized that studies that used matching might produce more conservative estimates of association). Univariate regression analyses showed that violation of HWE (β coefficient = 0.27 (0.06 to 0.48); HetP = 0.015, τ2 = 0.019), the mean age of cases (β = −0.01 (−0.02 to 0.0008); HetP = 0.066, τ2 = 0.024), and the method of case definition, clinically diagnosed CHD versus WHO criteria adjusted for other definitions (β = 0.26 (0.02 to 0.50); HetP = 0.038, τ2 = 0.020), were significant sources of heterogeneity among studies. The study size , the ethnicity , the male percentage in the study , blinded genotyping , sub-sample regenotyping , the source of controls , and matching  were not significant sources of heterogeneity among studies. Violation of HWE in multivariable regression analysis remained a statistically significant source of heterogeneity after adjustment for the effect of study size . Adding the mean age of cases and method of case definition to the model with violation of HWE decreased the τ2 value to 0.017 . It also showed that the effect of method of case definition on the variation among the studies was through the effect of the mean age on the heterogeneity and not as an independent factor. A model that included only violation of HWE and the mean age of cases reduced the τ2 value to 0.018 .First, an empty regression was run with only the log of the effect estimate of pooled studies under the additive model to determine the baseline value for τFirst, the influence of deviation from the HWE on effect estimates was examined by using HWE-deviated adjusted ORs. P-values equal to 0.066 and 0.074, respectively) suggested the presence of a publication bias. By using the trim and fill method, we showed that, if the publication bias was the only source of the funnel plot asymmetry, it needed seven more studies to be symmetrical  the absence of a biological effect, (ii) the presence of real genetic heterogeneity according to ethnic background, or (iii) failure to detect a small effect because the epidemiologic risk for an individual genetic variant is likely to be small and a large sample size is needed for adequate statistical power. It has been commonly proposed that, as well as a need for much larger and more rigorous studies those that are currently used, there is a greater need for international collaborations, particularly for a complex disease like CHD In this prospective study of healthy women aged 49 to 70 years, we investigated the relationship between the M235T polymorphism in the AGT genotypes in Hardy-Weinberg equilibrium. The limitations of this study were the relatively short period of follow-up and the small number of cases. Moreover, because this cohort was exclusively composed of Dutch women, these results cannot be generalized to men or other ethnic groups, for whom the rates of the events or the allele frequency are known to differ.In our study, the data collection was prospective, before the diagnosis of AMI or CHD and equal for all participants. This ensures that the cases and the randomly selected controls are comparable AGT gene in CHD risk. Although a pooled per-allele OR was suggestive of a modest increase in the risk of CHD of 1.08 , the robustness of this summary estimate is uncertain. First, in the pre-specified sub-groups analyses in the meta-analysis, larger studies, those with validated genotyping quality controls, and studies that used standardized criteria for case definition did not provide strong evidence for a positive statistically significant association between the M235T variant of the AGT gene and CHD risk. Second, the meta-regression analysis revealed that the HWE violation was a significant source of the moderate to large heterogeneity in the meta-analysis. Taking violation of HWE into account in the meta-analysis decreased the overall effect  or bias AGT gene and CHD was found, the relevance of this weakly positive overall association remains uncertain because it may be due to various residual biases. Moderate to large heterogeneity was identified between studies, and violation of HWE and the mean age of cases were statistically significant sources of the observed variation.In conclusion, the present meta-analysis, including our own data, indicated that, although a weak association between the M235T variant in the