This polymorphism was analyzed and correlated to total bilirubin and cholelithiasis in 270 age, gender, ethnically matched subjects  using PCR, dHPLC, fragment analysis and direct sequencing. Four genotypes of UGT1A1 were detected in this study . (TA)6/8 was found only in four individuals; hence it was not included in the analysis. There was a statistically significant association of genotypes with serum total bilirubin levels in both bTH and SCA groups (p<0.001). Subjects with (TA)7/7 had the highest total serum bilirubin level (178.7±3.5 µmole/l). A significant association was observed between allele (TA)7 and cholelithiasis development (p = 0.0001). The 40%, 67.5% and 100% of SCA with (TA)6/6, (TA)6/7 and (TA)7/7 respectively developed cholelithiasis and were subsequently cholecystectomized. Our results confirm UGT1A1 (TA)7 allele as one of the factors accounting for the hyperbilirubinemia and cholelithiasis observed in SCA and bTH.Present study was aimed to explore the effect of (TA) UGT1A1), the A1 isoform encoded by the bilirubin UDP glucuronosyltransferase 1 family (UGT1), catalyzes glucuronidation of bilirubin in humans through the formation of intermediate derivatives mono- and diglucoronides UGT1A1 enzyme to approximately 30% of normal levels Bilirubin is a toxic metabolite, predominantly resulting from the turnover of hemoglobin. Uridine diphosphate glucuronosyltransferase 1A1  7TAA polymorphism will probably increase the risk of developing cholelithiasis in such patients Beta Thalassemia major is a quantitative problem with a genetic defect that results in reduced rate of synthesis of the two beta globin chains causing severe anemia, often through mutations in regulatory genes. On the other hand, sickle-cell anemia (a hemoglobinopathy) is a qualitative problem of synthesis of an incorrectly functioning globin with mutation of the sixth amino acid valine to glutamine. The High levels of erythrocyte destruction in patients with SCA and to a lesser extend in bTH result in chronic hyperbilirubinemia. A significant proportion of patients are prone to cholelithiasis due to high biliary concentration of unconjugated bilirubin, which tends to coprecipitate with calcium in the gall bladder lumen. Cholelithiasis, by promoting cholecystitis and choledocholithiasis, is responsible for high levels of morbidity in hemoglobinopathy patients UGT1A1 (TA)n polymorphism in the Kuwaiti population except for one article screened the Kuwaiti G6PD patients for UGT1A1 (TA)n polymorphism without including healthy control No data have so far been reported about the prevalence of UGT1A1 (TA)n polymorphism in Kuwaiti population . Genotype (TA) 6/8 was detected only in 4 out of 236 tested cases; hence it was excluded from the analysis. Genotype (TA) 6/7 was predominant in both tested cases (≥63%) and controls (55%) while (TA) 7/7 was found to be rare (<6%) in both cohorts (UGT1A1 (TA)n polymorphism failed to show any significant difference between the tested subjects (p>0.05) with respect to age or gender.Screening region of interest revealed the existence of four genotypes of  cohorts . ObserveUGT1A1 (TA)n polymorphism with hyperbilirubinemia, serum total bilirubin of each tested subjects were detected. A significant association of average serum total bilirubin was observed with bTH and SCA subjects (p<0.05). Average serum total bilirubin was higher in SCA  than in bTH , when compared individually to healthy control (14.34±2.20 µmol/L) ((p = 0.0014) and 6/7 genotype (p = 0.0001) and SCA with 6/6, 6/7 and 7/7 genotype (p = 0.0001) when compared to individually to the healthy control with respective genotype. There was a significant association between the various genotypes of UGT1A1 (TA)n polymorphism and the serum total bilirubin levels in both bTH and SCA subjects (p<0.001), however no significant difference was observed based on gender.To evaluate the association of  µmol/L) . As showp<0.05 (UGT1A1 (TA)6\6 genotype developed gallstones, 76.5% heterozygotes (TA)6\7 and 100% homozygous SCA patients with UGT1A1 (TA)7\7 had cholelithiasis (All recruited SCA cases (104) underwent liver/biliary ultrasound scans and their data was available. A 67.3% of 104 of the tested SCA patients had gallstone disease. SCA patient having cholelithiasis had significantly higher serum total bilirubin (66.5±44.7 µmol/L), conjugated bilirubin (16.3±17.6 µmol/L) and unconjugated bilirubin concentrations (50.7±29.6 µmol/L) than the patients without cholelithiasis . No signp<0.05 . The freithiasis .p<0.05). An increased risk for cholelithiasis was observed among SCA when dominant genetic model was employed (6/6 versus (6/7+7/7) p = 0.0001, OR = 5.44, 95% CI 2.17–13.59), indicating the risk role of allele 7 in the development of gall stone.A significant association was observed between having allele (TA) 7 and the development of gallstone in SCA patients UGT1A1 promoter polymorphism and serum total bilirubin with tested subjects was further assessed using logistics regression by adjusting for factors such as age and gender  and SCA .Association of d gender . AnalysiUGT1A1 repeat, increases the risk of developing hyperbilirubinemia and cholelithiasis irrespective of disease status. The coinheritance of UGT1A1 polymorphism (7/7 or 6/7 repeat) and SCA was found to potentially increase the risk of developing cholelithiasis (OR = 5.44). We further assessed the global frequency of the genotype distribution of UGT1A1 promoter polymorphism according to the location on the various continents ((p = 0.448), the origin of all Arabs and other Arabic population revealed no significant difference. Similarly, no significant difference was found between the Kuwaitis and the Caucasian populations (p>0.05). A highly significant differences was found between Kuwaitis, Asians, Africans, southern Americans and the pacific islands (Papua New Guinea Tonga Fiji) populations p<0.05Our results in general sheds light on the fact that longer ntinents  [9], [11The theory of single gene disorders is no more accurate as the mutation in the globin genes alone is not sufficient to account for one of the major characteristics of RBC disorders such as bTH and SCA manifestations. There is a wide range of phenotypic expression of the disease, even in patients with identical hemoglobin genotypes from apparently similar environments UGT1A1 genotype to unconjugated hyperbilirubinemia and to the prevalence of cholelithiasis. Three  of the four known alleles in the UGT1A1 promoter region and four  of the six possible genotypes were retrieved. We compared patients with and without cholelithiasis and found that the frequency of UGT1A1 alleles (TA)6 was lower, and that of alleles (TA)7 was significantly higher in patients with cholelithiasis . These data suggest a possible relationship between the UGT1A1 locus and the occurrence of cholelithiasis in patients with SCA and bTH and most probably other hemoglobinopathies. Few studies have shown that allele (TA)7 is associated with high steady state concentrations of unconjugated bilirubin in serum and is a risk factor for cholelithiasis in adults with SCA or bTH. Furthermore, the importance of the pharmacogenomic effect of UGT1A1 polymorphism was shown in modulating the hematologic response to hydroxyurea treatment in SCA patients UGT1A1 genotypes identified and strongly suggested that UGT1A1 promoter polymorphism is a significant non-globin genetic modifier in these cases.Several risk factors for cholelithiasis have been identified in previous studies, one of which is the high total UGT1A1 gene Two global surveys showed a wide variation in the frequency of the number of (TA) repeats in the promoter region of the A total of 270 subjects were recruited in the study  after obtaining informed consent. Of these, 236 subjects who fit the selection criteria were carefully selected. All subjects were adult (>26 years of age) Kuwaiti Arabs with 1∶1.5 male to female ratios. The patients were regularly transfused with packed red cells every 4 weeks to maintain mean hemoglobin levels above 120 g/l, and were receiving regular iron chelating therapy (deferoxamine mesylate 40 mg/kg daily). Blood was withdrawn from the patients before receiving transfusion. Healthy Subjects were excluded if they had a past or present history of hepatic/hematological disease. None of the recruited bTH and SCA subjects had a history of primary hepatic disorders, excessive alcoholism, chronic use of medications or narcotics, nor had received any drug two weeks prior to our investigation. Clinical characteristics of each subject selected for the study is detailed in After an overnight fast, Blood and serum samples were collected from each subject in EDTA treated and plain tubes respectively. Plasma concentrations of total bilirubin were determined by diazo method at least three times within 6 months. Mean values were considered for the comparative study. Liver function tests were performed in the hospital laboratory by a standardized colorimetric procedure LXI, Beckman biochemical analyzer.5′-GAGGTTCTGGAAGTACTTTGC-3′ and the reverse primer 5′-CCAAGCATGCTCAGCCAG-3′ were used to amplify the region of interest in the 5′ region of the UGT1A1 promoter fragment of 409 bp. The PCR amplicons were subjected to dHPLC (denaturing high-performance liquid chromatography) to screen for the number of repeats in the TATA box. The presence of two well-resolved peaks reveals the heterozygous condition such as (TA)6/(TA)7. The presence of one peak is characteristic of the homozygous condition. To distinguish (TA)6 from (TA)7 homozygotes, each sample showing a single peak was mixed with (TA)6/(TA)6 control DNA under conditions allowing heteroduplex formation. The homozygous condition for (TA)7 was revealed by a double peak, whereas for (TA)6 homozygotes, there was no change in the chromatogram showing a single peak. Another screening method we used was the fragment analysis where the forward primer used was labeled with FAM fluoresce dye and amplicons were subjected to capillary electrophoresis. Genotyping results for 60 randomly selected samples (25% of tested cases) were further confirmed by sequencing using genetic analyzer ABI 3100 . The dHPLC results showed a 100% match with those obtained by direct sequencing and fragment analysis.DNA was extracted from the blood samples using the QIAamp Mini DNA extraction kit . The forward primer UGT1A1 promoter polymorphism were calculated manually for each subjects. Observed genotypes showed no significant deviation (p>0.05) from Hardy Weinberg equilibrium by Genepop software. Differences in the genotypic and the allelic frequency between the patients and controls were assessed by the chi-squared p value test. A p-value of <0.05 was considered to be statistically significant. Average serum total bilirubin is expressed as mean values ± Standard deviation (unless stated otherwise). Test for homogeneity (p<0.05) indicated asymmetric distribution, hence the total bilirubin data was log transformed and the statistical difference between the compared subgroups were assessed either by one-way ANOVA test or students t-test. Association of genotypes and total serum bilirubin level to disease status were assessed by binary logistic regression adjusting for factors such as gender and age.Data collection, data management and statistical analysis were performed with SPSS 19.0 . Allele and genotype frequencies of UGT1A1 (TA)n promoter polymorphism genotypes was determined for the first time in healthy Kuwaiti population, and is similar to frequencies observed in Caucasian populations. The extremely rare (TA)8 allele in Caucasians were also found in Kuwaitis. We further reveal that bTH and SCA patients (regardless of the age or gender) can be classified into three risk groups according to UGT1A1 genotype. Patients homozygous for (TA)7 are associated with a high frequency of cholelithiasis. UGT1A1 genotyping is therefore a potentially useful tool for identifying individuals with hemoglobinopathy at high risk of cholelithiasis and requiring close clinical monitoring.The frequency of


Infection with HIV may result in significant neuropsychological impairment, especially in late stage disease. To date, there have been no cohort studies of the impact of highly active anti-retroviral treatment (HAART) in South Africa where clade C HIV is predominant.Participants in the current study were recruited from a larger study of HIV-associated neurocognitive disorders (HAND) and included a group of individuals commencing HAART (n = 82). Baseline and one-year neuropsychological function was assessed using a detailed battery, and summary global deficit scores (GDS) obtained. Associations with change in GDS were calculated.p = 0.00 and 0.00 respectively). Participants with severe impairment at baseline improved significantly more than those with lesser degrees of impairment. Significant improvements were observed in the domains of attention, verbal fluency, motor function, and executive functions. There were unadjusted associations between GDS change and male gender, lower levels of education, baseline CD4 count and baseline GDS severity. In an adjusted model, only baseline GDS severity (p = 0.00) remained significant, with a lower level of education nearing significance (p = 0.05). The overall model was highly significant .Participants had a median CD4 cell count of 166 at baseline and 350 at follow-up. There were significant difference across groups of GDS severity at baseline with respect to level of education and GDS change at one year (In individuals in late stage HIV commencing HAART in South Africa, those with severe baseline neuropsychological impairment improved significantly more than those less impaired. While improvement across a number of neuropsychological domains was observed, high rates of impairment persisted.The effects of HAART and participant variables, such as test experience, require clarification. Studies with larger comparison groups, and where HIV disease characteristics are needed to establish whether the trends we identified are clinically meaningful. Neuropsychological impairments due to HIV infection of the CNS are detectable across all disease stages, but are more prevalent and marked in individuals with more severe HIV disease ,2. AlthoPersistent impairment may be the result of several interrelated factors, including host genetic vulnerability, ongoing low grade inflammation within the CNS and the effects of HAART itself . In addiSeveral published studies have reported on neuropsychological improvement following HAART initiation -13. The The impact of HAART on the CNS has been debated. A significant literature supports the contention that regimens of HAART that are able to penetrate the blood-brain barrier more readily, and in turn adequately suppress CSF viral load, are associated with better neuropsychological outcomes ,17,18. HThe impact of HAART in a prospective study in South Africa where clade C HIV is predominant has not been reported. In this study, we hypothesized that individuals initiating HAART would show improved neuropsychological function over one year. Moreover, we hypothesized that individuals with more severe disease at baseline would have worse neuropsychological outcomes at one year. Domain-specific changes were also explored.This study was conducted as part of a larger investigation of HIV-associated neurocognitive disorders (HAND) in Cape Town, South Africa that has been described previously . In summNormative data for neuropsychological testing of isiXhosa-speaking participants was obtained from 120 HIV- negative participants recruited from Voluntary Counseling and Testing services located at the same primary care clinics. Other than being HIV negative, as confirmed by a recent rapid HIV test and confirmatory serological test, inclusion and exclusion criteria were identical to the HIV-positive participants.The CD4 cell counts were extracted from the laboratory records, linked by the participants' clinic numbers. Clade sequencing was not available on this sample but more than 90% of infected individuals in the Cape Town area are infected with clade C virus . HepatitAll participants who met study criteria and agreed to participate provided written informed consent. Approval to conduct the study was obtained from the Research Ethics Committee of the Faculty of Health Sciences, University of Cape Town, and from the relevant health authorities.A neuropsychological test battery was administered to all participants to assess specific domains of neurocognitive function. The battery comprised tests of the domains: attention  and the Mental Control Test (MCT)), learning and memory  and the Brief Visuospatial Memory Test (BVMT)), psychomotor speed (Finger tapping (FT) and the Grooved Pegboard Test (GP)), psychomotor speed (Trail Making Test part A (TMTA), Color Trails Test 1(CT1) and Digit Symbol-Coding (DSC)), executive function (Color Trails Test 2 (CT2), Stroop Colour-Word test (SCW), Wisconsin Card-Sorting Test (WCST), and language (category fluency)).Raw data from the above neuropsychological test battery were converted to t-scores for each test using the demographically-adjusted normative data. From these, a deficit score for each test was obtained using the methods previously reported by Heaton and Carey ,23. The Data were analysed using STATA 11 . As data were not normally distributed across all variables, we calculated medians and interquartile ratios for the various cohort groups, and for the GDS severity group. Comparisons were made using Wilcoxon rank sum tests or Fisher Exact tests where appropriate. We generated line plots for the two time-points of the three GDS HAART-treated groups, and compared mean summary scores using Kruskal Wallis tests. Impairment in individual domains  were used to generate rates of impairment at baseline and follow-up. Differences in proportions across the time-points were calculated using McNemar's chi-square test. We then evaluated linear trends first using unadjusted models across the participants, with change in GDS between follow-up and baseline as the dependent variable. We included independent variables in a final multiple linear regression, if the simple model rendered a p-value of less than 0.1, or the variable was known to be strongly associated with neuropsychological test performance. The final model included gender, education, baseline and follow-up CD4 cell count, and baseline GDS.p = 0.14).A total of 166 participants were recruited into the cohort study. Of these, 58 were not re-assessed at one year for reasons including death (n = 9), migration (n = 6) and clinic loss-to-follow-up (n = 33). Of the 108 participants retained and who completed neuropsychological (NP) batteries at the two time points, 26 did not receive HAART and were not included in the cohort analysis. The three groups  were compared with respect to demographic and disease characteristics and there were no significant differences across groups (Table p = .00). There were no differences across groups for gender, home language, age, baseline CD4 count, duration of HAART or CPE. The groups differed for level of education, with the groups of increasing severity having a lower level of education (p = .00). The groups also differed with respect to change in GDS, with those more severely affected at baseline showing a greater degree of change in GDS (p = .00). The extent of GDS change is presented in Figure Demographic, disease and neuropsychological characteristics of participants initiating HAART across groups of baseline GDS severity are presented in Table p = .01), verbal fluency (p = .00), motor speed (p = .00) and executive functioning (p = .02). No improvement was noted for learning (p = .83) and psychomotor processing (p = .12). High rates of impairment persisted at one year (range 23-45%).The rates of impairment across neuropsychological domains at baseline and follow-up are shown in Figure p = .01), having a lower level of education (p = .00), baseline CD4 count (p = .04) and baseline GDS severity (p = .00) with GDS change (Table p = .00), while a lower level of education neared significance in the model (p = .05). The overall model was highly significant and provided a moderate level of predictive value . There was no effect of the duration of HAART or CPE on GDS change. In order to investigate potential associations between demographic and disease characteristics on baseline GDS, we examined unadjusted associations between these demographic and clinical variables and baseline GDs severity. Only a lower level of education was significantly associated with GDS .Potential predictors of GDS change were examined using a series of linear regression models. There were significant unadjusted associations between male gender . Only baseline GDS severity remained significant in adjusted models, while a lower level neared significance (p = 0.05). The strong trend to improvement in those most severely affected likely is explained by the range of possible improvement being wider; those with less impairment probably experienced a ceiling effect on their ability to improve. The improvement shown by individuals with less education in the adjusted model suggests that this effect was present together with the degree of impairment. This effect may be explained by participant characteristics such as test experience, whereby individuals with lower education benefitted more from the experience of being tested. In South Africa, the effects of quality of education, as opposed to levels of education, have been shown to exert effects in test settings [In the neuropsychology literature, improvement is usually associated with less baseline impairment . We notesettings .It is less well understood whether improvement in cohort studies is associated only with a HAART effect, or is also associated with practice effect, test familiarity or other participant characteristics, such as level and quality of education ,4. We reSeveral limitations of our study warrant discussion. We were able to retain a smaller sample of participants than originally recruited. The significant drop-out rate is due to a combination of factors, including large patient numbers at primary care clinics making tracking and retention by services difficult, mortality during early treatment, and migration of individuals. We also did not include additional groups of differently defined HIV positive participants who may have acted as controls. Such groups may have allowed for comparison of possible practice effects. These control groups may have included participants on stable HAART regimens, or those who were not. In the latter case, it would not be ethical to withhold treatment from individuals who met criteria for HAART. This group would therefore by definition have a different set of disease characteristics. We did not perform HIV subtype analysis on this sample, which would have allowed us to draw clearer conclusions about the putative role of clade in neuropathogenesis. Also, we were not able to obtain CSF in order to characterize intra-thecal viral load or inflammatory markers. The use of lumbar for research has not been approved widely in our setting. Lastly, we did not control for the range of baseline neuropsychological impairments we detected. The inclusion of a non-impaired group in measurement of change does affect group differences and outcomes.We believe that this study makes an important contribution to our understanding of the effect of HAART on neuropsychologically impaired individuals, in South Africa, where clade C HIV is highly prevalent. This benefit seemed to accrue most to individuals with more severe baseline impairment. Studies with larger comparison groups, and where HIV disease characteristics are similar, are needed to establish whether the trends we identified are clinically meaningful.The authors declare that they have no competing interests.JJ conceived and led the study, conducted data analysis and write the paper. JWT supervised neuropsychological assessments and scoring. JH assessed participants for neurologic disease. KT, RP and DJS participated in the design of the study and overall management. LM provided statistical consultancy. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/39/prepub


Although Iran is a high-risk region for esophageal squamous cell carcinoma (ESCC), dietary factors that may contribute to this high incidence have not been thoroughly studied. The aim of this study was to evaluate the effect of macronutrients, vitamins and minerals on the risk of ESCC.In this hospital-based case-control study, 47 cases with incident ESCC and 96 controls were interviewed and usual dietary intakes were collected using a validated food frequency questionnaire. Data were modeled through unconditional multiple logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI), controlling for age, sex, gastrointestinal reflux, body mass index, smoking history , physical activity, and education.ESCC cases consumed significantly more hot foods and beverages and fried and barbecued meals, compared to the controls (p < 0.05). After adjusting for potential confounders, the risk of ESCC increased significantly in the highest tertiles of saturated fat , cholesterol , discretionary calorie , sodium  and total fat intakes . In contrast, being in the highest tertile of carbohydrate, dietary fiber and (n-3) fatty acid intake reduced the ESCC risk by 78%, 71% and 68%, respectively. The most cancer-protective effect was observed for the combination of high folate and vitamin E intakes . Controls consumed 623.5 times higher selenium, 5.48 times as much β-carotene and 1.98 times as much α-tocopherol as the amount ESCC cases consumed.This study suggests that high intake of nutrients primarily found in plant-based foods is associated with a reduced esophageal cancer risk. Some nutrients such as folate, vitamin E and selenium might play major roles in the etiology of ESCC and their status may eventually be used as an epidemiological marker for esophageal cancer in Iran, and perhaps other high-risk regions. Esophageal squamous cell carcinoma (ESCC) is the sixth most common cancer in the world and the fourth most common in the developing countries ,2 with aThe alarmingly high incidence of ESCC and its equal sex distribution in Iran highlights the likelihood of a very strong environmental risk factor as the main culprit . Gross nWhile previous studies have mostly focused on food items in relation to ESCC risk, studying macro- and micronutrients could offer advantages mainly through providing better understanding of underlying mechanisms of disease . AccordiThis hospital-based case-control study was conducted in Kurdistan, a high-risk province of Iran. Cases were patients aged 40-75 years, who visited major general hospitals and had incident histologically-confirmed ESCC. Cases did not have history of carcinoma of other sites and were interviewed within 6 months after their ESCC was diagnosed. Controls were chosen from individuals admitted to the same hospitals as the cases for a wide spectrum of acute non-neoplastic diseases including traumas , surgical conditions , non-traumatic orthopedic conditions  and miscellaneous diseases . Cases and controls were frequency-matched according to the sex and age (5-year groups).Generally, 50 patients with ESCC and 100 hospital controls were interviewed face-to-face by professionally-trained interviewers using structured pre-tested questionnaires which evaluated socio-demographic characteristics , smoking history , eating habits , medical history, medication use ), gastroesophageal reflux disease (GERD) symptoms (heartburn and acid regurgitation) and familial cancer history . QuestioWeight was measured with subjects clothed minimally, standing on digital scales  without shoes and was recorded to the nearest 100 grams. Height was measured using a non-stretch tape meter fixed to a wall with subjects standing without shoes and was recorded to the nearest 0.5 cm. Body mass index (BMI) was then calculated by dividing weight in kilograms by square of height in meters. Physical activity was measured using a validated questionnaire comprising of different metabolic equivalent (MET) categories , and it Two patients were excluded from the analyses since their reported energy intakes were below or above 3 standard deviations from the mean, indicating errors in reporting dietary intakes . We furtA validated semi-quantitative food frequency questionnaire (FFQ) was used by trained dietitians in face-to-face interviews to evaluate the usual dietary intakes of participants during the previous year ,50. HabiPortion sizes of consumed foods were specified according to the US Department of Agriculture (USDA) standard portion sizes  and were then converted to grams. When using the USDA portion sizes was impossible, household measures  were used alternatively . PatientAverage daily intake of each food item was calculated by multiplying the consumption frequency of each food by its standard item-specific portion size from the exchange list; these scores were then summed to estimate nutrient intakes. The estimates of nutrient intakes in the present paper are derived from the dietary sources alone. Since Iranian food composition table (FCT) is incomplete and provides data only on a few nutrients , analyseFor ordinal variables, chi-square test or Fisher's exact test and for continuous variables, Kruskal-Wallis test or t-student test were used to compare case and control groups. Macro- and micronutrient intakes were adjusted for total energy consumption using the residual method as suggested by Willet and Stampfer . Energy-2kg/m2), smoking status (never/former/current), smoking intensity and duration , physical activity (MET) (light/heavy), and education level .For comparison purposes, we calculated a base regression model and a fully-adjusted model for each analysis. The base model was adjusted for the matching variables, i.e. age (years) and sex , which are controlled for automatically by design. The fully-adjusted model, on the other hand, included the following covariates: age (years), sex , GERD symptoms (yes/no), BMI  and also the risk factor (nutrient intakes). Other potential confounders such as monthly family income, place of residence , familial cancer history, cooking method and food and beverage temperature did not alter the main effect estimates for dietary factors and therefore were not included in the final models.As a basis for trend testing, scores were constructed from the categorized variables as successive integers and were used in further analyses. All statistical tests were carried out using the Statistical Package for Social Sciences, version 16  and a two-tailed P-value of <0.05 was considered statistically significantThe present study was approved by all regional ethics committees in Iran and also the "Ethics Committee of the National Nutrition and Food Technology Research Institute", Shahid Beheshti University of Medical Sciences, Tehran, Iran. Written informed consents were taken from each subject prior to the interviews.Table The calorie-adjusted mean values for selected macronutrients and relative risk estimates of ESCC by tertiles of macronutrient intake residuals are presented in Table In the fully-adjusted model, those in the highest tertile of SFA intake had 2.88 times higher ESCC risk , followed by those in the highest intake tertile of cholesterol , discretionary calorie  and total fat intake . On the other hand, being in the highest tertiles of carbohydrate, dietary fiber and (n-3) fatty acid reduced the risk of ESCC by 78%, 71% and 68%, respectively. In the preliminary age- and sex- adjusted analysis , a positive association also emerged with an increased protein intake, which was not significant in the fully-adjusted model.6, folate, vitamin B12, vitamin C, iron, calcium, phosphorus, methionine and selenium were significantly higher among controls compared to ESCC cases (p < 0.05), while average adjusted sodium intake was significantly higher among cases compared to the controls ( p < 0.001)  and riboflavin intakes   fatty acids and among micronutrients, higher intakes of folate, vitamin E and selenium have the most protective effects against ESCC in a high-risk population in Iran. An increased ESCC risk estimate was observed among those with highest intakes of SFA, cholesterol, discretionary calorie, sodium and total fat. Most importantly, being in the highest tertile of joint folate and vitamin E intake was associated with 98% reduction in the ESCC risk.This is the first study in a high-risk population to evaluate the impact of a wide range of macronutrients, minerals and vitamins on risk of ESCC. Similar to previous case-control studies, we found a decreased ESCC risk associated with higher intakes of nutrients with plant origin and an increased risk for intake of several nutrients found primarily in animal-based foods ,29,58-61Epidemiological studies have shown a positive association between total fat, cholesterol and SFA intakes with ESCC and esophageal adenocarcinoma risk ,57,64-67Findings of previous studies have been inconclusive regarding the role of protein intake in esophageal cancer risk with some classic studies suggesting an inverse relationship ,69. We oHigh intake of dietary fiber in the present study decreased ESCC risk by about 70%. Although few studies have questioned the role of dietary fiber in cancer protection -74, mostIt has been suggested that the cancer-protective effects of fruits and vegetables intake is mediated through their several antioxidants and dietary components, such as folate, vitamin A, β-carotene, vitamin C and dietary fiber ,69,80-83Epidemiological evidence regarding the role of folate intake in ESCC risk are scanty ,67,84,852, B6, B12 and methionine have major roles in one-carbon metabolism. Previous studies in Iran have reported inadequate riboflavin intakes among patients with esophageal cancer [6 intakes among ESCC cases also, might leads to chromosome breakage [Similar to folate, vitamin Bl cancer . This isl cancer ,69,82,91breakage , defectibreakage ,67,85.12 intake and ESCC risk. However, it has been suggested that the positive relationship between vitamin B12 and EC risk observed in some studies [12 is derived exclusively from animal sources and hence may be simply a marker for consumption of animal protein and other factors or nutrients in these foods [9 and B12 intakes, compared to those living in the low risk areas [12 deficiency disorders (e.g. pernicious anemia) are at greater risk of EC [12 intake and ESCC risk is not yet clear and evidence in this regard is lacking. In the present study, high methionine intake was inversely associated with ESCC risk, which might be explained through its involvement in SAM production, which is necessary for retaining folate in body [In this study, we failed to observe a significant association between vitamin B studies , could bse foods . It has sk areas  and thossk of EC ,95. Over in body Higher intakes of vitamin E and vitamin D in this study were associated with about 90% and 70% ESCC risk reduction, respectively. The finding of a strong inverse association between ESCC with vitamin D intake in this study is consistent with some studies ,98, althDietary antioxidants  have been shown to decrease the EC risk ,98,101 tVitamin A and β-carotene were not significantly associated with ESCC risk in our population, which is in line with several studies ,98,106 aIn the present study, higher sodium intake was associated with almost 50% increase in the ESCC risk. Some epidemiological studies have suggested a role for higher salt intake in carcinogenesis. Although salt is not a carcinogen per se, it acts as an irritant to the esophageal protective mucosal layer, which results in inflammatory regenerative response, increased DNA synthesis and cell proliferation  and may Deficiency of several vitamins/minerals has been associated with higher EC incidence, with the most pronounced effect observed in the developing countries . PreviouThis study has several limitations. Firstly, as with other case-control studies, recall bias and selection bias were inevitable. In case-control studies, there is the possibility that cases may recall their diets differently after a cancer diagnosis. However, our participants were generally of low literacy and socioeconomic status with little knowledge about the role of diet and nutrients in the cancer risk, which should have reduced the possibility of recall bias. Moreover, using hospital controls and administering validated FFQs by trained interviewers in a hospital setting might have further reduced the recall bias and improved comparability of information of cases and controls ,116. WitNot having data on participants' alcohol consumption was yet another barrier. Our subjects refrained from reporting their alcohol intake due to the fact that consuming alcohol is legally prohibited in Iran ,118. In The third limitation is the possibility of some micronutrient misclassification due to not having data on supplement use. However, it has been suggested that dietary supplements and fortified foods, mask the beneficial effect of food intakes in reducing the cancer risk  and someAnother limitation of the present study was using a semi-quantitative FFQ, which despite its common use for characterizing the habitual dietary intakes, is well-recognized for its weakness in quantification of nutrient intakes . Using aAnother drawback of this study was using nutrient values in the statistical analyses without directly referring to the foods which contributed most to the nutrient intake and its variation. According to Willet, an optimal approach to epidemiologic analysis is to employ both foods and nutrients to represent diets ; in thisAnother potential source of error is that several naturally continuous variables  were categorized for the purpose of analysis, which might have increased the possibility of residual confounding and decreased the precision and power of the study. However, we compensated for this limitation by choosing categories with multiple sufficiently narrow intervals to decrease the residual confounding and heterogeneity of subjects within intervals.2, B6, B9 and B12. This complexity added to the problems of using estimated intake of nutrients obtained at one point in time must be considered when interpreting the results of this study. Small sample size is also a limitation which might have resulted in unstable results and extreme relative risk estimates observed in some of the subgroups, although this is one of the largest sample series in an Iranian population and a number of strong consistent findings have emerged from this sample [Additionally, availability of B-vitamins is influenced by diet, supplement use, alcohol consumption and generic polymorphism and B-vitamins are all involved in one-carbon metabolism which requires vitamin Bs sample ,50.This study has several strengths. Firstly, eliminating information bias associated with use of proxy data enabled us to consider numerous potential cofounders. Detailed assessment and adjustment for several important confounders and total energy intake are other important aspects of this study. We attempted to reduce the measurement error and false-positive effect by calculating nutrient intake residuals standardized for total energy intake rather than reporting absolute nutrient values. This further accounted for the confounding effect of total energy intake on nutrient intake estimates .In the present study, a validated FFQ was used which provided subjects with the option of answering in terms of day, week or month which enhanced reporting precision considering the fact that frequency of consumption is a truly continuous variable . In addiFinally, evaluating the nutrient-cancer relationship in a population without mandatory nutrient fortification, where supplement use is uncommon and nutrient intakes are low has enabled us to capture the association of nutrients and ESCC more clearly compared to previous studies ; as suchIn conclusion, findings of the present study suggest that higher SFA, cholesterol, discretionary calorie, sodium and fat intakes significantly increase the risk of ESCC, whereas dietary antioxidants, especially folate, vitamin E and selenium could prevent the damage to the esophagus caused by oxidative stress, even if consumed in moderate amounts. Our results suggest that dietary patterns rich in carbohydrate, dietary fiber and (n-3) PUFAs along with high physical activity and low consumption of hot foods and beverages and fried/barbecued meals should be promoted in order to prevent ESCC in Iranian population. However, prospective cohort studies that evaluate diet before the cancer diagnosis, as well as interventional studies that address nutrient deficiencies are warranted to clarify whether changes in dietary practices and/or vitamin and mineral supplementation can reduce the incidence of ESCC in Iran.Overall, this study has mainly the character of a pilot hypothesis-generating study conducted in a region with low prevalence of alcohol consumption and high rates of ESCC, allowing a further search for risk factors of this cancer site. It is recommended that these findings should be replicated, particularly in comparable samples  to identify dietary factors that could substitute for the dominating role of alcohol and smoking for cancers of the upper gastrointestinal tract seen in many Western societies.The authors declare that they have no competing interests.M.J contributed to the conception and design of this study, data analysis, interpretation and drafting of the manuscript. B.R assisted in conception, design, data acquisition, analysis and drafting of manuscript. B.H collected the data and participated in designing the study. M.J. critically reviewed and helped to draft the manuscript. C.G provided methodological feedback and gave the final approval to the manuscript to be published. All authors have read and approved the final manuscript.Calorie-adjusted mean values among esophageal cancer cases and controls, and range of macronutrient intakes in tertile categories of selected macronutrients in a case-control study in Iran. The file contains information about the mean macronutrient intakes among cases and controlsClick here for fileCalorie-adjusted mean values among esophageal cancer cases and controls, and range of micronutrient intakes in tertile categories of selected micronutrients in a case-control study in Iran. The file contains information about the mean micronutrient intakes among cases and controlsClick here for file


FOXE1 as a thyroid cancer (TC) susceptibility gene in populations of European descent. After that, a number of studies reported that the rs965513, rs1867277, and rs71369530 polymorphism in FOXE1 has been implicated in TC risk. However, the causal variants remain unknown. To derive a more precise estimation of the relationship, a meta-analysis of 9,828 TC cases and 109,995 controls from 14 case–control studies was performed. Overall, significant results were observed for rs965513 , rs1867277  and rs71369530  polymorphism. In the subgroup analysis by ethnicity, we found that rs965513 polymorphism confer high risk for Caucasians with per-allele OR of 1.80  compared to East Asians of 1.35 . There was strong evidence of heterogeneity, which largely disappeared after stratification by ethnicity. In the subgroup analysis by sample size, and study design, significantly increased risks were found for the polymorphism. In conclusion, this meta-analysis demonstrated that common variations of FOXE1 are a risk factor associated with increased TC susceptibility.Forkhead box E1 encodes the transcription factor FOXE1 (or TTF-2), which together with Homeobox protein NKX2-1, PAX8 and HHEX, are pivotal proteins required for thyroid gland formation, differentiation and function. Recently, genome-wide association studies have identified  Thyroid cancer (TC) is the most common endocrine malignancy, and accounts for 1% of all neoplasias FOXE1 at chromosome 9q22, has been identified as a new hotspot for thyroid cancer susceptibility by a recent GWA study FOXE1 possesses a polymorphic polyalanine tract (rs71369530) just distal to its DNA-binding domain, with 11–22 alanine residues reported, although FOXE1 14Ala and FOXE1 16Ala account for greater than 98% of reported alleles FOXE1 gene (rs1867277) was positively associated with sporadic thyroid cancer susceptibility. Over the past few years, these polymorphisms  in the FOXE1 region and thyroid cancer risk have been independently replicated by subsequent studies. As stated by McClellan and King, many if not most of the genetic polymorphisms that are reported to be associated with common disorders in GWA studies are factually spurious associations caused by subtle differences in ancestry between the populations being studied (population stratification) FOXE1 and thyroid cancer.Recently, spectacular advance was made in identifying susceptible genes involved in TC through genome-wide association strategy (GWAS) among European descent FOXE1 gene were sought by computer-based searches from databases including Pubmed, SCOPUS, ISI web of knowledge, Embase, Cochrane databases and CNKI  without language restriction. Search term combinations were keywords relating to the FOXE1 gene  in combination with words related to thyroid cancer . We replaced one term each time until all possible combination mode were searched to avoid any missing literature. The titles and abstracts of potential articles were screened to determine their relevance, and any clearly irrelevant studies were excluded. The full texts of the remaining articles were read to determine whether they contained information on the topic of interest. Furthermore, reference lists of primary studies and review articles were also reviewed by a manual search to identify additional relevant publications.Epidemiological genetic association studies published before the end of Nov. 2013 on thyroid cancer and polymorphism in the The included studies have to meet the following criteria: (1) evaluation of at least one of those three polymorphisms  and thyroid cancer risks using case–control or cohort design, (2) original papers containing independent data, (3) identification of thyroid cancer patients was confirmed histologically or pathologically, (4) genotype distribution information or odds ratio (OR) with its 95% confidence interval (CI) and P-value, and (5) genotype distribution of control group must be consistent with Hardy–Weinberg equilibrium (HWE). The major reasons for exclusion of studies were (1) overlapping data, (2) case-only studies, and (3) review articles.Information was carefully extracted from all eligible publications independently by the two authors according to the inclusion criteria listed above. For each included study, the following information was extracted from each report according to a fixed protocol: first author, publication year, definition and numbers of cases and controls, frequency of genotypes, age, sex, ethnicity, HWE status, source of control, histological subtypes and genotyping method. Review reports from the two were then compared to identify any inconsistency, and differences were resolved by further discussion among all authors. Studies with different ethnic groups were considered as individual studies for our analyses. Meanwhile, different case–control groups in one study were considered as independent studies. The instrument “Extended-quality score” was used to assess the quality of association studies FOXE1 polymorphism and thyroid cancer risks. The per-allele OR of the risk allele was estimated. Then, we estimated the risks of the heterozygous and homozygous genotypes on TC compared with the wild-type homozygote 2 based Q-statistic and I2 test 2 values <25% correspond to no or little heterogeneity, values 25–50% correspond to moderate heterogeneity, and values >50% correspond to strong heterogeneity between studies. Random-effects and fixed-effect summary measures were calculated as inverse-variance-weighted average of the log odds ratio Crude ORs with the corresponding 95% CIs were used to assess the strength of the association between the The combined search yielded 96 references. −5; −5) and homozygous for the risk A allele  when compared with the wild type genotype.There was a wide variation in the A allele frequency of the rs965513 polymorphism among the controls across different ethnicities, ranging from 0.06 to 0.44 . For Eas−5; heterozygous: OR = 2.60, 95% CI: 2.06–3.02, P<10−5; homozygote: OR = 3.36, 95% CI: 2.75–4.51, P<10−5). Similar significant associations were also observed for East Asians with per-allele OR of 1.35 . Analysis restricted to the 8 studies with at least 500 cases, which should be less prone to selective publication than smaller studies, yielded an OR of 1.75 . As for smaller studies, the summary OR of rs965513-A variant for TC was 1.67 . By considering study design subgroups, the OR was 1.75  in GWAS compared to 1.70  in candidate gene studies .In view of significant heterogeneity and to seek for its potential sources, we performed a panel of subgroup analyses on ethnicity and sample size. When studies were stratified for ethnicity, significant risks were found among Caucasians in all comparisons . In meta-regression analysis, sample size (P = 0.68), and study design (P = 0.89), did not significantly explained such heterogeneity. By contrast, ethnicity (P = 0.001) was significantly correlated with the magnitude of the genetic effect.−5; −5) and 2.63 , respectively.The A allele frequency in Caucasians was 0.40 (95% CI: 0.38–0.43). Using random effect model, the per-allele overall OR of the A variant for TC was 1.64  alleles, 16-Ala was the alleles showing higher frequencies in cases than controls. The overall per-allele OR of the 16-Ala variant for total TC was 2.01  was significantly associated with increased risk of TC, particularly in Caucasian population. As studies among other ethnic populations are currently limited, further studies including a wider spectrum of subjects to investigate the role of those variants in other populations will be needed. Besides, future studies are recommended to identify the possible gene-gene and gene-environmental interactions in this association.Despite these limitations, this meta-analysis suggests that the three common variations on Figure S1Study selection process.(TIF)Click here for additional data file.Figure S2FOXE1 rs965513 among controls stratified by ethnicity. The “Frequencies of the risk alleles of (TIF)Click here for additional data file.Figure S3FOXE1 rs1867277 polymorphism and thyroid cancer risk.Forest plot for association of (TIF)Click here for additional data file.Figure S4FOXE1 polyAla variant (71369530) and thyroid cancer risk.Forest plot for association of (TIF)Click here for additional data file.Figure S5FOXE1 rs965513 polymorphism and thyroid cancer risk.Result of sensitivity analyses for (TIF)Click here for additional data file.Figure S6FOXE1 rs1867277 polymorphism and thyroid cancer risk.Result of sensitivity analyses for (TIF)Click here for additional data file.Figure S7FOXE1 rs71369530 polymorphism and thyroid cancer risk.Result of sensitivity analyses for (TIF)Click here for additional data file.Figure S8FOXE1 rs965513 polymorphism and thyroid cancer.Begg's funnel plot for publication bias in studies on (TIF)Click here for additional data file.Figure S9FOXE1 rs1867277 polymorphism and thyroid cancer.Begg's funnel plot for publication bias in studies on (TIF)Click here for additional data file.Figure S10FOXE1 rs71369530 polymorphism and thyroid cancer.Begg's funnel plot for publication bias in studies on (TIF)Click here for additional data file.Checklist S1(DOC)Click here for additional data file.


Background. Many studies have reported an association between tea drinking and Parkinson's disease (PD). Our purpose is to summarize the available information and evaluate the risk of PD associated with tea drinking. Methods. We searched all publications in English language on the association of tea drinking and PD risk published up to December 2010. The pooled analysis was performed with Review Manager 5.0. Results. In total, eight articles including 1418 cases and 4250 controls were included in the meta-analysis. The pooled odds ratio (95% CI) was 0.85 (0.74–0.98), which suggests the protective effect of tea drinking in PD risks. Moreover, the summary OR  for drinkers of ≤1 cup of tea per day versus nonconsumers and that  for drinkers of >1 cups of tea per day versus nonconsumers showed that there was not an apparent dose-response relationship. No indication for publication bias was found. Conclusions. This meta-analysis showed that tea drinking can lower the risk of PD, while no apparent dose-response relationship was found. Further effort is needed to fully understand the mechanism underlying the beneficial effect of tea consumption in lowering PD risk. Parkinson's disease (PD) is a common neurodegenerative disorder, and, with the accelerated aging of human society, the prevalence of PD is expected to rise steadily in future , 2. DespWe identified procurable published studies in English through a computerized MEDLINE search from 1966 to December 2010 and also the relational document links. We used the following medical subject headings and free text words to search the PubMed database: (TEA) AND (PARKINSON OR PD) AND (CASE-CONTROL OR CASE-REFERENT OR RETROSPECTIVE OR COHORT OR FOLLOW-UP OR INCIDENCE OR PROSPECTIVE OR EPIDEMIOLOG). We also examined conference proceedings; the references in the articles retrieved through the computerized search. The identified studies must meet the following criteria: (i) the original data includes the number of PD cases studied and the odds ratio (OR) or relative risk (RR), (ii) its corresponding 95% confidence interval (CI) is for highest versus non-/lowest level of tea intake, and (iii) these studies must define the outcome of interest as incident PD based on clearly stated diagnostic criteria or identified through diagnostic codes with additional confirmation. In total, eight published articles were included in the meta-analysis –15. The P  value >0.10, it means that the study does not have statistical significance. We used the fixed effect model to calculate the summary OR and its 95% CI across homogeneous studies and used the random effect model to calculate the summary OR and its 95% CI across heterogeneous studies. Egger's weighted regression method was employed to evaluate P value for publication bias. Finally, stratified analyses were conducted to examine the differences by cups of drinking. Statistical computation was performed using the Review Manager 5.0 statistical software.We extracted information from each paper including first author, year, and country of publication, the number of cases and controls, the adjusted RRs or ORs, and 95% CI. We examined possible heterogeneity in the identified result. If the P = 0.04). The analyses compared exposed with unexposed for tea drinking, and the overall summary OR based on all studies indicated that tea drinking can protect PD  with eight cases.The eight case-control studies (including 1418 cases and 4250 controls) which met the eligibility criteria were conducted in 5 countries between 1966 and 2010. Among the eight studies that included in our present analysis, three reported the results for drinkers of ≤1 and >1 cup of tea per day versus non-consumers. , 16, 18.As we know, PD is a common neurodegenerative disorder in middle- and old-aged crowd. The etiology of PD has long been thought to involve many factors, and there were five major hypotheses regarding the pathogenesis of PD, such as the theory of genetic factors, the theory of environmental factors, immunity doctrine, apoptosis theory, and the oxidative stress theory –22. In oTea is one of the most ancient and popular beverages consumed around the world. There are many important substances in tea leaves, such as polyphenols, methylxanthine, caffeine, different lipids, amino acids, mineral substances, and volatile compounds. The demonstrated health benefits of tea include weight loss, preventing heart disease and cancer, and so forth . For exaIn recent years, there were more and more studies devoted to exploring the effects of tea consumption on PD risk. Many studies reported that tea drinking can lower the risk of PD , 12–15, Moreover, according to the present meta-analysis, we did not find an apparent dose-response relationship as expected. It may arise from the following factors: (i) black tea and green tea differ markedly in the nature of their polyphenols , 38, whiThere were heterogeneity and limitations in our study. Firstly, the population studied in the included references was small and their location was not widespread, which make us unable to perform analysis on race. Secondly, other factors with possible influence on disease progression such as anti-Parkinsonian drugs, food supplements, or alcohol consumption were not considered. Thirdly, there were only few studies reporting stratified results according to specific types of tea (black or green) and the gender of the population studied. If more data is available in future for performing stratified analyses according to the types of tea, through comparing the components of the specific types of tea, we can identify the most important compounds, which will be of significance to the prevention and treatment of PD.To summarize, our findings indicate that tea drinking can protect against PD, which leads human to value the impact of lifestyle on health. Despite the mechanisms behind this association should be multiple, in view of the widely accepted pathogenic role of oxidative stress in PD and the well-documented antioxidant potential of bioactive tea components, the antioxidant activity is expected to be an important aspect. Further effort is encouraged to fully understand the comprehensive mechanism underlying the beneficial effect of tea consumption in lowering PD risk.


The onset and progression of breast cancer (BC) is influenced by many factors, including the single nucleotide polymorphism (SNP) rs13281615 at 8q24. However, studies of the potential association between rs13281615 at 8q24 and risk of BC have given inconsistent results. We performed a meta-analysis to address this controversy.PubMed, EMBASE and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. Two curators independently extracted data, and odds ratios (ORs) with 95% confidence intervals (95% CIs) were calculated to assess the strength of the association between rs13281615 at 8q24 and risk of BC.P<0.001; G-allele vs. A-allele, OR 1.10, 95% CI 1.06–1.14, P<0.001; GG vs. AA, OR 1.20, 95% CI 1.12–1.29, P<0.001). Conversely, the AA genotype is significantly associated with decreased risk of BC .Fourteen studies are included in the meta-analysis, involving 44,283 cases  and 55,756 controls . The GG and G-allele genotypes of rs13281615 at 8q24 are significantly associated with increased risk of BC (GG vs. AG+AA, OR 1.13, 95% CI 1.08–1.19, G-allele genotypes of rs13281615 at 8q24 polymorphism are a risk factor for developing BC, while the AA genotype is a protective factor. Further large and well-designed studies are required to confirm this conclusion. Breast cancer (BC) is one of the most prevalent invasive cancers and the second leading global cause of cancer-related deaths among women BRCA1 and BRCA2, have low mutation rates and therefore explain only a small fraction of breast cancers in the general population Many researchers have concluded that BC is the cumulative result of multiple environmental factors and genetic alterations Two large-scale genome-wide association studies identified several common polymorphisms that may be linked to susceptibility to BC Ever since three studies PubMed, EMBASE and the Chinese National Knowledge Infrastructure (CNKI) databases were searched through December 2012 for case-control studies about 8q24 rs13281615 SNP and BC risk. The following search terms were used: “8q24,” “rs13281615,” “SNP,” “single nucleotide polymorphism,” “polymorphism,” “mutation,” “variant,” “BC,” “breast neoplasm” and “breast cancer.” Reference lists in each identified article were also searched manually to identify additional eligible studies.To be included in the meta-analysis, studies had to (1) assess the association between the 8q24 rs13281615 SNP and risk of BC occurrence, (2) use a case-control design and (3) provide sufficient data for estimating odds ratios (ORs) with 95% confidence intervals (CIs). In the case of multiple studies by the same researchers involving the same or overlapping data sets, we selected the most recent study with the largest number of participants.Two curators (W-FG and J-HZ) independently extracted information from included studies. Disagreement was resolved by discussion between the two authors. The following data were extracted: first author’s family name, year of publication, country of origin, source of controls, total numbers of cases and controls, Hardy-Weinberg equilibrium (HWE) of controls and the frequency of rs13281615 genotypes at 8q24 in cases and controls.All statistical tests were performed using Review Manager 5.1.4 software. The strength of association between the 8q24 rs13281615 SNP and BC risk was assessed by calculating ORs with 95% CIs based on the genotype frequencies in cases and controls. Subgroup analysis was performed based on ethnicity, categorized as Chinese or mixed (predominantly Caucasian).P<0.05 defined as the significance threshold. Meta-analysis was conducted using the random-effects model, except when P>0.10 for the Q-test, indicating lack of heterogeneity among studies; in this case, the fixed-effects model was used. Publication bias was assessed by visual inspection of Begg’s funnel plots. HWE in the control group was assessed using the asymptotic test, with P<0.05 considered significant.The significance of pooled ORs was determined using the Z-test, with P>0.05) in all but three studies Database searches through December 2012 revealed 512 potentially relevant publications about rs13281615 at 8q24 and risk of BC . ScreeniP<0.001; I2 = 51%)     . In the 2 = 42%) . In the 2 = 56%) . Most im2 = 67%) .P<0.001, I2 = 58%). In the homozygote comparison model, the GG genotype was associated with higher risk of BC . In the dominant genetic comparison model, the AA genotype was associated with lower risk of BC . By allelic comparison, the G-allele was also associated with lower risk of BC .After stratifying the data by ethnicity and applying the recessive comparison model to the mixed population, we found the GG genotype to be associated with higher risk of BC . The AA genotype was associated with lower risk of BC based on the random-effects model .Analysis of the Chinese population in four studies Sensitivity analysis was carried out by excluding one study with large heterogeneity Begg’s funnel plots were calculated to assess publication bias for reported comparisons of 8q24 rs13281615 SNP and risk of BC. The shape of the funnel plot seemed asymmetrical for GG vs. AG+AA, AA vs. AG+AA, GG vs. AA, and G-allele vs. A-allele, suggesting the presence of publication bias .As with other malignancies, the pathogenesis of BC involves environmental factors, molecular signaling pathways and host genetic factors. Genome-wide association studies conducted since 2007 have identified several genetic loci associated with susceptibility to BC. Some studies have reported an association between 8q24 rs13281615 SNP and BC risk, while others have found no such association. The most likely reason for the inconsistencies among these studies is that they involve relatively small samples. To conduct association studies with larger numbers of participants, we conducted a meta-analysis of published studies. Our results for the total population suggest increased BC risk for subjects carrying the G-allele of rs13281615 at 8q24.Subgroup analysis by ethnicity allowed us to look for potential ethnic differences in the association. In the Chinese population, the G-allele was associated with increased risk of BC based on allelic contrast, recessive contrast and homozygote comparison. Similarly, for the mixed population (>95% Caucasian), the G-allele was also associated with increased risk of BC. In both the Chinese and mixed populations, the AA genotype of rs13281615 was associated with lower risk of BC. Sensitivity analysis did not alter these results.Several studies included in the meta-analysis demonstrate that rs13281615 polymorphisms can interact with environmental factors to modulate BC risk Incidence of BC and associated mortality differ widely across ethnicities P value of HWE of three included studies There are several limitations in this meta-analysis. First, the controls were not uniformly defined. Although all controls were healthy populations, it is possible that some of them were community-based, while others were hospital-based. The Our meta-analysis suggests that the 8q24 rs13281615 SNP affects risk of developing BC: the G-allele increases susceptibility to BC, while the A-allele protects against it. These findings add BC to the list of diseases for which the 8q24 rs13281615 SNP has been implicated as a susceptibility factor, together with prostate cancer This meta-analysis is guided by the PRISMA statement .Figure S1Forest plots describing the association of the 8q24 rs13281615 single nucleotide polymorphism with risk of developing breast cancer (AA vs. AG+GG).(TIF)Click here for additional data file.Figure S2Forest plots describing the association of the 8q24 rs13281615 single nucleotide polymorphism with risk of developing breast cancer (GG vs. AG+AA).(TIF)Click here for additional data file.Figure S3Forest plots describing the association of the 8q24 rs13281615 single nucleotide polymorphism with risk of developing breast cancer (GG vs. AA).(TIF)Click here for additional data file.Figure S4Begg’s funnel plots to examine publication bias for reported comparisons of the 8q24 rs13281615 single nucleotide polymorphism and risk of BC. Data were plotted using pseudo 95% confidence limits. SE, standard error.(TIF)Click here for additional data file.Table S1Sensitivity analysis results with random-effect model and fixed-effect model.(DOC)Click here for additional data file.Checklist S1PRISMA 2009 Checklist.(DOC)Click here for additional data file.


To determine the association between the number of thymine-adenine (TA)n dinucleotide repeats in the promoter region of the gene coding for the estrogen receptor alpha (ESR1) and the prevalence of lone atrial fibrillation (AF) in men.We conducted a case-control study involving 89 men with lone AF and 166 healthy male controls. The ESR1 genotype was established by polymerase chain reaction and capillary electrophoresis. To assess the association of ESR1 genotype with AF, logistic regression models were built with AF as outcome.P = 0.010). After adjustment for other factors, a unit-increase in (TA)n repeat number was associated with a significantly greater likelihood of AF .Men with lone AF had significantly greater number of (TA)n repeats of single alleles than controls n repeats in the promoter region of ESR1 is associated with a significantly increased likelihood of lone atrial fibrillation in men. Atrial fibrillation (AF) is considered the most common sustained arrhythmia of clinical importance . Even ifGenetic determinants of AF have been explored using different methods, ranging from candidate gene studies investigating rare mutations  with altLess attention, however, has been paid to the association of AF and nucleotide polymorphisms of cardiac sexual steroid receptors. Sexual steroid hormones exert complex genomic and non-genomic cardiac effects through corresponding receptors . The genThis case-control study included 89 male patients with lone AF and otherwise structurally and functionally normal hearts and 166 healthy male participants without any known disease. All participants were of Caucasian origin recruited from the city of Zagreb and the wider region and were included in the study as they appeared in our hospital or outpatient clinic from January 2010 to December 2012 . InformeThe main inclusion criterion for the AF group was electrocardiographically (ECG) documented AF in the absence of known etiological factors for AF  . All AF The control group consisted of volunteers from the preventive health check program with healthy hearts and normal electrocardiograms. On physical examination they were found to have no signs or symptoms of any disease, as people with not normal test results were excluded from the study  (Supplementary Table)Height was measured using a Harpender stadiometer and weight on a digital scale without shoes. Body mass index (BMI) was calculated as the weight in kilograms divided by the square of height in meters. Blood pressure was measured in a sitting position using a standard analog sphygmomanometer. Mean arterial pressure (MAP) was calculated by the formula: systolic blood pressure (SBP) × 1/3 + diastolic blood pressure (DBP) × 2/3. The serum estradiol level was assessed by a competitive immunoassay technique .The transthoracic echocardiogram was performed using a GE Vivid 7 echocardiograph . All examinations were performed by cardiologists with accreditation in transthoracic echo-cardiography by the European Society of Cardiology. The images were later reanalyzed using the GE Health Care EchoPac Dimension software, PC version 108.1.3 .Genomic DNA was extracted from peripheral blood leucocytes using the Nucleon II DNA extraction kit  according to the manufacturer’s instructions. Analysis was performed within one week after the blood sample was obtained.Dinucleotide polymorphism of ESR1 was analyzed using polymerase chain reaction (PCR) amplification with labeled primer 5′-6-FAM-GAC GCA TGA TAT ACT TCA CC-3′ and 5′ - GCA GAA TCA AAT ATC CAG ATG-3′. PCR was performed with 25 cycles consisting of 2 minutes at 94°C, 1 minute at 58°C, and 1 minute at 72°C, followed by 30 minutes at 60°C after the last cycle. The alleles were size-separated by capillary gel electrophoresis using Gene Scan Fragment Analysis Software 4.0 . In short, 1 μL of the product was diluted with 12 μL of deionized formamide containing 0.5 μL GeneScan-500 ROX internal lane standard for sizing DNA fragments. Capillary electrophoresis was then carried out using ABI PRISM 310 Genetic Analyzer and POP-6 , which had been created for applications requiring high resolution under denaturing electrophoretic conditions .2 test for categorical variables. To assess the association of ESR genotype with AF, binary logistic regression models were built with AF as outcome. Model 1 was unadjusted, whereas Model 2 was adjusted for age, MAP, and serum estradiol concentration. Likelihood ratio test was used to determine the significance of odds ratios. For all analyses, we used statistical package STATISTICA, version 9.1. (www.statsoft.com) and a double-sided P < 0.05 was considered significant.Data are shown as mean values with the standard deviation (SD) for continuous variables and absolute numbers with prevalence (%) for categorical variables. Departure from normal distribution was initially evaluated for all continuous variables by Kolmogorov-Smirnov test and graphically. Mann-Whitney U test and Kruskal-Wallis test were conducted to compare the differences between cases and controls for continuous variables and χThere was no significant difference in clinical, echocardiographycal, and biochemical characteristics between cases and control group . We obseP = 0.003, χ2 = 11.7, χ2 test), while the prevalence of the SL combination  and the SS combination  were lower in the AF group.The ESR1 gene promoter TA repeats were classified as short  and long , corresponding to their bimodal distribution and in accordance with previous studies on cardiovascular diseases and non-cardiovascular diseases (P = 0.010). We also created  five groups of examinees based on the sum of (TA)n repeats of both of their alleles to be used as predictors and observed greater prevalence of AF with a greater sum of (TA)n repeats in a dose-dependent manner , while serum estradiol concentration did not differ significantly between the groups  n repeats in single alleles between the AF and control group and found a significant difference  .P = 0.002). When adjustment was made for age, blood pressure and serum estradiol concentration were added (Model 2), the observed association remained significant and its magnitude did not change.To test whether the observed relationship was confounded by already known risk factors of AF, logistic regression was performed . In ModeOur results suggest a positive association between the number of (TA)n repeats in the promoter region of ESR1 and the prevalence of lone AF in men. The significance of the observed relationship after adjustment for age, serum estradiol, and blood pressure indicates that the effect of (TA)n is independent of these putative confounders.The basic role of estrogens in both sexes is modulation of immune responses, oxidative stress, inflammation, cellular growth, proliferation and apoptosis, and affecting the central nervous, cardiovascular, and skeletal systems ,19. EvidThe proposed mechanisms by which sex steroids affect cardiac electrophysiology include their effect on nuclear and extranuclear receptors in cardiomyocites ,21,22, mESR1 repeats are only one of many ESR1 polymorphisms  and theiElectrophysiological research on ESR1 polymorphisms has so far been mostly related to ventricular rather than to atrial arrhythmias. To our knowledge, this is the second report of the effect of the (TA)n repeat ESR1 polymorphism on atrial fibrillation, the first one being a small pilot-study that suggested a correlation between homozygosity for alleles with a larger number of (TA)n repeats and AF . StudiesOur study has several limitations. We were able to analyze only the (TA)n repeat polymorphism. It is possible that other polymorphisms of the same gene or polymorphisms of the gene encoding the estrogen receptor β could also be associated with lone AF. The study was limited to male participants so the results may not be applicable to women.Our results indicate that a greater number of (TA)n repeats in the promoter region of ESR1 is associated with an incremental increase in the likelihood of lone AF in men. This result could give new insights into the role of steroid hormones in the pathogenesis of AF. Large-scale studies conducted in different populations are required to more comprehensively and reliably assess the relationship of (TA)n length with the quantity and quality of ER-α transcripts, and the occurrence of AF.


Galicia, in northwest Spain, experiences warm summers and winters. However, the higher relative humidity that prevails the whole year through and the location of the summer hot points are related to real weather heat stroke in the hottest season. However, Planet Global Heating was recently analyzed for the climate in Galicia. Climate change was found to be able to trigger effects that involve a new situation with new potential regions of risk. In this paper, 50 weather stations were selected to sample the weather conditions in this humid region, over the last 10 years. From these results, new regions with a potential for heat stroke risk in the next 20 years were identified using the humidex index.Results reveal that during the last 10 years, the winter season presents more comfortable conditions, whereas the summer season presents the highest humidex value. Further, the higher relative humidity throughout the whole year reveals that the humidex index clearly depends upon the outdoor temperature.Global Planet Heating shows a definite effect on the outdoor comfort conditions reaching unbearable degrees in the really hottest zones. Therefore, this effect will clearly influence tourism and risk prevention strategies in these areas. European weather changes commence from the Atlantic, and thereafter, the events that occur there determine, to a large degree, the weather and the climate of Europe. Galicia, located in the northwest of Spain enjoys mild climate, mainly influenced by the Azores anticyclone, Iceland depression, and central Europe thermal anticyclone. The Azores anticyclone controls the movement of the depression zones towards the Galician coast, which is influenced by the annual revolution of the Earth around the sun.However, it is noteworthy that the extreme climatic conditions during the summer months pose a significant public health threat. For example, the unfavourable weather conditions were related  to the dAn important, and most often neglected, parameter in the relationship between weather and mortality is climate change. In this sense, the recent papers -6 showedOnce the possible reasons for global climatic changes happen and their consequences are explained, their effects on future tourism  and subsHowever, the experimental inductive (field research of micro-climatic variables and subjective answers) and deductive (simulation of predictive models) as in ,12 methoClimatic data was sampled in 50 weather stations located in the important places in Galicia, as indicated in Figure Climatic change models were obtained from the references  after a Where,t is the mean seasonal temperature increment (°C)∆τ is the time (years)Once these models are obtained, they can be used to predict the climatic changes in Galicia for the next few years. The year 2030 was chosen due to the great interest in the effect of climate change on the occupation of different parts of Galicia in order to develop future action plans, like civil constructions and architectural building designs, for population protection. From the perspective of preventing heat-related illnesses and deaths, some European cities developed hot-weather response plans .In some recent research works, two different heat indices were compared; the first index being the National Weather Service (NWS) Heat Index (HI) derived from previous databases and the second being humidex, a Canadian innovation. This index is employed by the Meteorological Services Canadian (MSC) . In 1979An extremely high humidex reading is defined as one that crosses 40, as shown in Table 2) higher than HI, as we can see in Equation 5.Despite its limitations, humidex remains a useful, and hence popular, means of determining how hot it actually feels outside. Both indices present advantages; for example, the HI presents a linear relationship dependency with the predicted rectal temperature. Humidex thus presents an exponential model which, despite its greater complexity, shows a better average correlation coefficient (RWhere T is the temperature in Celsius and e is the vapor pressure in millibars (mb).Therefore, and in accordance with the Meteorological Services Canada (MSC), humidex was selected.Figures In this paper, 50 weather stations were selected to sample the weather conditions in this humid region over the last 10 years. As mentioned earlier, Galicia, located in the northwest of Spain, experiences warm summers with mean temperatures of 20°C, and winters with 6°C, as seen in Figure This effect is due to the increase in the humidex with temperature and relative humidity, as shown in Figure Once this relationship is analyzed for the particular Galicia mean conditions, Galician temperature distribution should be analyzed in July for each region as in Figure From a statistical study of the instantaneous values of the meteorological station, the region with the highest mean maximum temperature was identified. This region (see Figure To analyze the future climatic change effect, it is necessary to study the weather changes over the last 10 years. The results revealed that during the last 10 years, the winter season presents the better comfort conditions with a mean humidex of 7, while the summer season presents the highest humidex with a mean value of 18. Therefore, due to the higher relative humidity the whole year round, this humidex value reveals a clear dependence on the outdoor temperature.From the data sampled and with the predicted change in the climatic conditions for these regions, new zones with the potential of heat stroke risk in the next 20 years can be identified. Thus, Global Planet Heating presents a clear picture of the outdoor comfort conditions reaching unbearable values. An increase in the mean Galician temperature for next 20 years has been predicted to be 1.35°C during the summer and winter seasons; however, the more extreme increment of 1.89°C will be expected during the spring season [From these predicted temperature changes it is possible to estimate the seasonal humidex for the next 20 years, as shown in Figure On analyzing the instantaneous effect of climate change in Ourense, the hottest point on the comfort conditions it is evident that maximum outdoor temperatures with values near 40°C are prevalent towards the end of July, as seen in Figure On analyzing the increase in temperature due to climate change and extrapolating for the instantaneous outdoor conditions of the year 2030, we obtained the data shown in Figure These temperatures and relative humidity were sampled in the climatic stations located near the town, and therefore, these values will reveal an increase in the zones identified within the town. This is the “urban heat island effect” which causes individuals living in cities to be susceptible to a high degree of risk of death when the temperature and humidity are high as compared with those living in the suburban and rural areas . TherefoFuture risk prevention in populations due to heat stroke must consider these predictions as a good index to improve the future planning and town design. For example, although thermal comfort is difficult to achieve passively, shading is one of the most significant properties that emerges with increased vegetation, thus mitigating the heat stress . HoweverFinally, research work in the future is necessary to define a better calibration methodology  of the iGalicia experiences warm summers with mean temperatures of 20°C, and winters with 6°C. Despite these warm temperatures, the higher relative humidity of 70% that prevails throughout the year indicates a high mean humidex value.In this study, 50 weather stations were selected to sample the weather conditions in this humid region over the last 10 years to identify the zones experiencing the highest mean maximum temperature. However, Planet Global Heating was analyzed in the recent studies for Galician climate causing a rise in the average temperature that involves a new situation with new potential regions of risk.From the data sampled and with the predicted changes in climatic conditions for these regions, the new regions with the potential of heat stroke risk in the next 20 years can be predicted. From these predicted temperature changes, the humidex for the next 20 years for each season can be estimated.Specifically, an increase of 1.35°C in the mean Galician temperature for next 20 years during the summer and winter seasons was predicted; however, the more extreme increase of 1.89°C will be expected during the spring season. The lowest outdoor temperature increment is expected during the autumn season, with an increment of 0.81°C. Therefore, the humidex will show a mean increment from 7 to 10 and from 18 to 21, in the winter and summer seasons, respectively. On further analysis, the temperature increase due to climate change, extrapolating for the instantaneous outdoor conditions of the year 2030, makes it evident that this humidex will experience an increase in its maximum value of 43, which is related to dangerous ambience at the hottest point.Finally, the humidex values are expected to reach the maximum supportable levels, and therefore, this fact must be taken into account in future town design. Further, future risk prevention in the population due to heat stroke must consider these predictions as an index to improve the future realizations. As an example of this methodology, the high risk regions will need to change their building construction materials and design characteristics to prevent risky situations, as well as to reduce their energy consumption.The authors declare that they have no competing interests.JO Design of the study and humidex implementation. AC weather modelling and data analysis. ARF Design of the study and data analysis. GR Design of the study and climate change analysis. All authors read and approved the final manuscript.


To evaluate the association between diagnosis of Parkinson disease (PD) and risk factors or early symptoms amenable to population-based screening.A systematic review and meta-analysis of risk factors for PD.The strongest associations with later diagnosis of PD were found for having a first-degree or any relative with PD  or any relative with tremor , constipation , or lack of smoking history , each at least doubling the risk of PD. Further positive significant associations were found for history of anxiety or depression, pesticide exposure, head injury, rural living, beta-blockers, farming occupation, and well-water drinking, and negative significant associations were found for coffee drinking, hypertension, nonsteroidal anti-inflammatory drugs, calcium channel blockers, and alcohol, but not for diabetes mellitus, cancer, oral contraceptive pill use, surgical menopause, hormone replacement therapy, statins, acetaminophen/paracetamol, aspirin, tea drinking, history of general anesthesia, or gastric ulcers. In the systematic review, additional associations included negative associations with raised serum urate, and single studies or studies with conflicting results.The strongest risk factors associated with later PD diagnosis are having a family history of PD or tremor, a history of constipation, and lack of smoking history. Further factors also but less strongly contribute to risk of PD diagnosis or, as some premotor symptoms, require further standardized studies to demonstrate the magnitude of risk associated with them. ANN NEUROL 2012 Idiopathic Parkinson disease (PD) has a prevalence of approximately 1% in individuals aged >65 years.We followed PRISMA 2009 guidelines for systematic review and meta-analysis and the Cochrane Collaboration definition of both terms.Published studies were included if they fulfilled the following criteria: (1) assessed at least 1 risk factor or early nonmotor symptom preceding a subsequent diagnosis of PD; (2) reported original data on relative risks (RRs) or odds ratios (ORs) from cohorts representative of the general population or case–control studies with cases defined as patients diagnosed with PD; and (3) reported data that could be easily obtained in a primary care environment, that is, those factors that could be determined through questionnaires or widely available blood tests.We excluded review articles, editorials, commentaries, hypothesis papers, letters that reported no new data, meta-analyses, and abstracts. We excluded studies that (1) reported on treatment and management of PD, (2) considered associations with established PD , (3) reported factors not easily ascertainable in the primary care setting , (4) studied young onset PD only, (5) did not use a control group or provide adequate details of the control group , (6) used blood relatives as the control group, (7) were twin studies, (8) were genetic studies or laboratory studies not used widely, (9) reported on the same risk factor in a common study population , (10) reported on a disease other than PD, or (11) reported measures other than OR/RR or an equivalent  or from which an OR could not be calculated. If there was disagreement between authors , the articles were discussed in further detail until an agreement was reached.Study characteristics, a risk estimate of the main study finding, and secondary findings were extracted for all eligible studies using a standardized template. Only factors for which a significant association was reported in at least 1 study were included. We included risk factors according to binary measurements . Data that reported exposures as quartiles or quintiles where the lowest exposure quartile was equal to zero were converted to binary terms. We excluded associations reported with medical conditions, drugs or toxins known to cause symptomatic Parkinsonism such as antipsychotics or carbon monoxide poisoning. We did not include studies that reported associations with dementia occurring before onset of PD, as these cases would not fulfill current criteria for PD and might include cases of dementia with Lewy bodies.2 statistic and, where statistically significant heterogeneity was found (p < 0.05), the random effects model was used to combine results.Where a factor of interest was reported by 2 or more studies in a consistent manner, these were combined in a meta-analysis; first separately for case–control and cohort studies (given that cohort studies are less subject to bias), and second for all studies together (considering odds ratios from case–control studies to be estimates of relative risks) to generate a pooled effect size and 95% confidence interval (CI) for each factor. Heterogeneity between studies was assessed using the IThe literature search yielded 3,856 English language articles, of which 202 were eligible for inclusion in the systematic review and 173 of these in the meta-analysis . The reaSummary results for positive and negative associations with PD found in the meta-analysis and those where no association was found are shown in p = 0.017), coffee drinking (p = 0.002), pesticides (p < 0.001), hormone replacement therapy (p = 0.016), statins (p = 0.038), any family history of PD (p = 0.011), family history of tremor (p = 0.009), and well water drinking (p = 0.005). Using the trim and fill method to account for the bias had no effect on the summary estimate for ever smoking, coffee, hormone replacement therapy or statins, but did diminish the summary estimates for pesticides , any family history of PD , family history of tremor , and well water drinking ; however, the conclusion that these are statistically significant risk factors for PD was not altered.According to the Egger test, there was evidence of publication bias for the factors ever smoking  consumption also reducing risk. For smoking, the effect is strongest in current smokers and weakest in past smokers (56% for current smokers and 22% for past smokers), but the association remains significant in all. The nature of this association is still poorly understood, and cannot be explained simply by selection bias and confounding.38Pesticide and environmental toxin exposure have been often implicated in PD causation.Head injury may carry increased risk of PD, particularly in those with repeated head injury and pugilist's encephalopathy.This systematic review reaffirms the proposed early nonmotor symptoms, which may predate the diagnosis of PD by several years. Constipation and mood disorders appear to approximately double an individual's risk of subsequent PD in the meta-analysis. Both constipation and mood disorders have been suggested to correlate with brainstem involvement that, together with olfactory bulb involvement, occurs early in PD and later spreads to the substantia nigra and to the cortex.48Hypertension was associated with reduced risk of PD, but the role of selective mortality in these individuals has not been studied. The combined analysis of studies of calcium channel blockers suggests a mild but significant overall reduction of PD risk. Beta blockers, on the other hand, were associated with increased PD risk. This could, however, potentially be explained by trials of beta blockers in those with seemingly isolated tremor. A history of diabetes mellitus did not significantly alter risk; however, further studies may be necessary, given that the case–control studies showed a statistically significant decrease in risk, whereas the cohort studies, showed a statistically significant increase in risk. Statins showed a trend of reduction in risk of subsequent PD in this meta-analysis, which may be due to a protective role through reduction of oxidative stress.NSAIDs are associated with risk reduction by approximately 17%. A similar finding has recently been published in a Cochrane review of NSAIDs preceding PD diagnosis.53We did not find any association of subsequent PD with the oral contraceptive pill, surgical menopause, or hormone replacement therapy, although further studies on the latter are needed, given that case–control studies showed a statistically significant decrease in risk, whereas cohort studies showed a statistically significant increase in risk. There was no significant association of PD and preceding cancer. Although we did not include melanoma as a specific search term, a recent meta-analysis suggests a positive association of established PD with melanoma, but no association if melanoma precedes a diagnosis of PD.54Helicobacter pylori infection has been suggested as a risk factor for PD in recent years, but there are no studies that specifically look at H. pylori and subsequent risk of PD. A surrogate marker for this could be gastric ulceration, but in our meta-analysis there was no association with prior gastric ulcer and subsequent PD diagnosis.Age and gender were not included in this review, as they are mainly reported in uncontrolled prevalence studies. Thus, although some of the studies included in the analysis reported risk associated with these variables, a combined risk estimate is unlikely to be representative of the full published literature on these factors. Nevertheless, the increased prevalence rate with increasing age and the higher prevalence in men are well recognized, and reporting is therefore usually stratified by age and gender.55We restricted our search to articles written in English, and therefore reports written in other languages are not included in the analysis. Due to the large number of studies used and the heterogeneity of methods and reported findings, we selected variables according to the main criterion that they can be screened for on a population basis. Therefore, risk factors and early symptoms not easily screened for would not have been included. Not all studies reported estimates of risk adjusted for confounders . Where such adjustments have been made, we have included these data in the analysis.Risk factors were dichotomized, which ignores dose effects. However, an essential feature of this analysis was to define factors that could be defined in binary terms, to allow them to be employed in combination for large-scale screening.2 = 50–75%), and in 4 there was high heterogeneity (I2 > 75%). This is expected, because of the differences between individual studies in, for example, case ascertainment, study population characteristics, exposure measurement, and whether crude or adjusted risk estimates were reported. For this reason, the pooled estimates should be interpreted cautiously, in particular for those instances of high heterogeneity .Statistically significant heterogeneity was found in the majority (24 of 30) of meta-analyses performed, similar to what has previously been reported.This is the first comprehensive systematic review and meta-analysis taking into account all risk factors for PD suitable for screening in primary care. This report allows comparative assessment of the evidence for, and magnitude of association for, the numerous proposed risk and early disease factors of PD, and helps to understand better their contribution to the risk of PD.