The meta-analysis involved 1092 EH patients and 1152 controls. The pooled odds ratios (ORs) and their corresponding 95% confidence interval (CI) were calculated by a fixed or random effect model.The present meta-analysis is performed to investigate the relationship between the TNFα G308A gene polymorphism and EH. Electronic databases were searched and seven separate studies on the association of the TNFα G308A gene polymorphism and EH was found in an allelic genetic model , a recessive genetic model , and a homozygote model . No significant association between them was detected in both a dominant genetic model  or a heterozygote genetic model .A significant relationship between the TNFα G308A gene polymorphism is associated with EH susceptibility.The  There are about one billion essential hypertension (EH) patients in the world, and the current EH morbidity in China is about 11.8%. Statistical data from the Ministry of Health of the People's Republic of China indicate that the incidence of cerebrovascular diseases increases up to two million cases each year, and 60% of which are associated with EH. Among EH sufferers, 1.5 million die of stroke every year. The Framingham heart study has shown that in the United States, the EH risk in 55-year-old normotension individuals is 90% throughout their lives.EH is a common disease influenced by polygenic and environmental factors. Finding a new gene associated with EH will help clarify the pathogenesis of EH and provide a new therapeutic strategy. The pathogenesis of EH is not limited to cardiovascular areas and is also related to immunology as well as heredity Tumor necrosis factor-alpha (TNF α), the inflammation promoter, is secreted primarily by mononuclear phagocyte cells. TNF α induces endothelial cells to secrete vasoactive substances via the autocrine or paracrine pattern, which leads to vasorelaxation or vasoconstriction, and ultimately, to the regulation of blood pressure (BP) TNF α gene is located in the major histocompatibility complex III region on chromosome 6p21.3. Recent research has revealed that TNF α gene polymorphisms are mostly focused on the probable influence of the promoter district on the expression of the TNFα gene. The TNFα gene polymorphism is also involved in infectious diseases, metabolic syndrome, stroke, hyperuricemia, and so on th TNF α gene promoter can influence TNF α expression at the transcription level TNFα G308A gene polymorphism and EH is relatively limited, the results are still controversial In 2003, Bogdanski et al. TNFα G308A gene polymorphism and EH  EH diagnosis in accordance with the EH diagnosis requirements of the World Health Organization established in 1999, c) subjects had a systolic BP≥140 mmHg or a diastolic BP≥90 mmHg, and d) subjects had no secondary hypertension.The inclusion criteria were as follows: a) evaluation of the All data were collected according to a standard protocol. Studies that were repeated, of poor research quality, did not meet the inclusion criteria, and provided little information or insufficient data were excluded. TNFα G308A gene polymorphism and EH was compared by the odds ratio (OR) and the corresponding 95% confidence interval (CI) between the EH and control groups. The heterogeneity of between-studies was determined by the Chi-square-based Q-test, and the significance was fixed at the level P<0.05 I2 was also calculated to evaluate the variation caused by the heterogeneity. A high value of I2 indicated a higher probability of the existence of heterogeneity . If there was heterogeneity among the studies, the random-effects model was used to estimate the pooled OR by the DerSimonian and Laird method P<0.05. Fisher's exact test was used to assess the Hardy-Weinberg equilibrium and the significance was set at P<0.05. Potential publication bias was estimated using a funnel plot. Egger's linear regression test was used to evaluate the funnel plot asymmetry on the natural logarithmic scale of the OR  The association between the TNFα G308A gene polymorphism. One paper was excluded because it deviated from the Hardy-Weinberg equilibrium , a recessive genetic model , and a homozygote model . The results from the three genetic models were positive and the heterogenetity between the invidual studies did not exist which indicated that there was an intensively positive association between TNFα 308A allele and EH risk.A significant association between the P = 0.06, Pheterogeneity = 0.003) or a heterozygote genetic model  .The publication bias of the included studies was assessed by the funnel plot and Egger's test. The funnel plot showed no apparent evidence of publication bias . There wTNFα G308A gene polymorphism and EH in an allelic genetic model (OR: 1.45), a recessive genetic model (OR: 3.181), and a homozygote genetic model (OR: 3.454). The A allele of the TNFα G308A gene may be the susceptibility gene for EH. This result was the strength of this meta-analysis.The present study suggested a significant association between the EH is one of the principally harmful disorders to public health. EH morbidity gradually increases annually, and the EH onset age becomes younger on the average. Apart from heredity, dietary, psychological, and neuroendocrinal factors, inflammation also plays an important part in the EH mechanism TNFα can enter the blood and exhibit hormone-like effects. Increased TNFα exerts an immediate cytotoxic effect by facilitating neutrophilic granulocyte degranulation, oxidative metabolism, and accelerated lipid peroxidation. It can destroy the structural integrity and function of endothelial cells, causing an imbalance in endothelial cells that secrete active substances. Decreased synthesis and release of vasodilators as nitric oxide and increased secretion of vasoconstrictors as endothelin and prostaglandin indirectly expedite vasoconstriction as well as elevate peripheral resistance and BP. A low perfusion condition caused by the capillary contraction of the EH target organ also ensues TNFα is a sensitive inflammation indicator. It has a direct injury effect on endothelial cells depending on the dosage. At low concentration levels, TNF α is a short-distance regulatory factor that can induce target cells to produce cytokines and antibodies. At high concentration levels, TNFα G308A gene increases TNFα transcription activity and the serum TNF α level. This allele has also been associated with obesity, type 2 diabetes mellitus, coronary artery disease, serum C reactive protein, and insulin resistance The A allele of the The divergence among the different research results may be due to the following limitations:TNF-alpha may play a role in modulating BP and low-density-lipoprotein cholesterol in Japanese women. However, in 2002, Krikovszky et al. TNF-alpha-308A allele carrier state appears to be associated with the low systolic and diastolic BP values in diabetic adolescents in Hungary. In 2007, Yoo et al. TNF-alpha-308G allele carrier is associated with increased EH risk in Koreans. In China, Guo TNF-alpha-308A allele. Sookoian et al. also confirmed this point in Argentina Ethnic differences. In 2001, Ito et al. Geographic isolation. In the seven studies included in the current meta-analysis, the pressure of natural selection caused an idiopathic gene polymorphism distribution structure. This phenomenon can be attributed to the fact that the research subjects in the seven studies came from different districts.Sample size. In the current meta-analysis, the total sample size was relatively small and the results may be unclear. Increased sample size is recommended in further research.TNFα G308A gene may be predisposed to EH. Due to the aforementioned limitations, further investigations should be conducted to verify these findings.Finally, the present meta-analysis suggests that the frequency distribution of the A allele of the Supporting Information S1PRISMA 2009 Checklist.(DOC)Click here for additional data file.Supporting Information S2PRISMA 2009 Flow Diagram.(DOC)Click here for additional data file.


This study aimed to determine risk factors associated with congenital toxoplasmosis in Minas Gerais which is the second largest Brazilian State based on number of inhabitants, and its territorial extension is larger than that of France. Methods: Population-based case-control study to assess the association between congenital toxoplasmosis and maternal exposure to infection risk factors. The study included mothers/children participating in the Minas Gerais Newborn Screening Program. The cases consisted of 175 mothers of infected children, and the controls consisted of 278 mothers of children without suspected infection. The associations were assessed through binomial logistic regression with The variables associated with lower probability of toxoplasmosis were: older mother age , higher level of education , access to potable water , and home with flush toilet . The variables associated with higher probability of infection were: cats in the neighborhood , owning or visiting homes with domestic cats , handling the soil , and eating fresh meat not previously frozen . After stratification according region of residence , home with flush toilet and consumption of treated water were protective against the disease only in the rural stratum.Toxoplasma gondii, the predominating risk factors were those related to the ingestion of oocysts. It is expected that these results will contribute to development of a program for prevention of congenital toxoplasmosis adapted to the reality of the population of Minas Gerais. The differences between populations living in rural and urban areas regarding the main risk factors for toxoplasmosis point to the need of considering regional specificities in planning strategies to control congenital toxoplasmosis.In Minas Gerais, congenital toxoplasmosis has been associated with poor socioeconomic conditions. Considering maternal exposure to sources of  Toxoplasma gondii. Its prevalence varies according to geographic region, as well as socioeconomic and cultural factors Toxoplasmosis is a disease caused by the ubiquitous protozoan The main social impact of toxoplasmosis in humans is associated with vertical infection. In this situation, the parasite is capable of causing severe disease, with short and/or long term sequelae. Infected newborns may be asymptomatic at birth or display a wide array of signs/symptoms, from unspecific systemic involvement to severe neurological and ocular damage, as well as hearing impairment There are three major strategies to control congenital toxoplasmosis: prenatal and newborn screening and, health education. The educational approach is the only measure truly capable of preventing infection among pregnant women. However, educational measures will have an impact on the women's behavior only if they are adequately sensitized to the need of changing habits As the sources of infection are multiple and vary across regions Congenital toxoplasmosis was screened in the State using dried blood collected from the participants of the Newborn Screening Program of the State of Minas Gerais (PETN-MG) over a period of seven months. The screening pointed to high prevalence of the infection, 13 cases in every 10 000 newborns, and a regional social inequity regarding prevalence rates of the infection Given the importance of toxoplasmosis infection in this Brazilian context this study aims at identifying the main risk factors associated with congenital toxoplasmosis in the State of Minas Gerais and contributing to developing a prevention program adapted to the regional context.o. 69/2007); Centro de Treinamento e Referência em Doenças Infecciosas Orestes Diniz (no. 16/2007), and UFMG Ethics Committee (ETIC 510/07). Written informed consent was obtained from the parents or guardians of all participating children.The study protocol followed the tenets of the Declaration of Helsinki. The study received approval in all required instances: Universidade Federal de Minas Gerais (UFMG), School of Medicine, Departments of Pediatrics . The study included newborns participating in the PETN-MG.The study was conducted in Minas Gerais, a state in Southeastern Brazil with 853 municipalities and 19,597,330 inhabitants in total, spread over an area of 586,520 kmT.gondii IgM tests of dried blood collected in filter paper from newborns. Confirmative plasma serum tests were run on cases with positive or undetermined results – anti-T.gondii IgG and IgM antibodies (children/mothers), anti-T. gondii IgA antibodies (children). The infants were referred to the outpatient clinic of the UFMG University Hospital for clinical examination and complementary investigations. All of them received treatment as soon as the diagnosis was confirmed The study included as cases the mothers of newborns confirmed with congenital toxoplasmosis  accordinT.gondii IgM and/or IgA and positive IgG until age of 6 months; (2) negative anti-T.gondii IgM/IgA and positive IgG associated with retinochoroidal lesions within the first six months of life; (3) persistence of positive anti-T.gondii IgG results until age of 12 months The criteria for confirmed congenital toxoplasmosis were: (1) positive anti-Once the number of cases had been defined (n = 175), the number of controls was estimated with reference to the 5% level of significance (α), and the test power (1 − β) of 80%, based on reports in the literature.The population-based controls were selected among mothers of infants included in the aforementioned program in the year 2011. These newborns were not tested for toxoplasmosis, as the program screening had not included this disease in the period under scrutiny. From 141 out of 853 municipalities in the State that reported cases of congenital toxoplasmosis from November 2006 through May 2007, four strata were creating according to performance in the Minas Gerais State Social Responsibility Index, a socioeconomic indicator created by the Center for Public Policy Studies at Fundação João Pinheiro with a view to depicting the level of development of the municipalities in the State Mothers of infants with confirmed congenital toxoplasmosis who brought their children to outpatient clinic of HC-UFMG and could be interviewed were included as cases.The controls included a randomly selected sample of mothers/children previously invited to participate who went to the health center in the assigned municipalities. The mothers of infants randomly selected who missed their interviews were replaced by mothers of children aged up to seven months who went to the health center for regular examination, immunization or collection of dried blood with filter paper within the scope of PETN-MG. Since these newborns were not tested for toxoplasmosis, as the program screening had not included this disease in the period under scrutiny, a careful investigation was performed in order to exclude mothers/children suspected of acute gestational toxoplasmosis/congenital toxoplasmosis, through mother inquiry, prenatal appointments and children health reports.Interviews with the cases were carried out from November 2006 through May 2007, and with the controls from May through August 2011 . Upon inThe mothers of the controls were invited for interviews at the main health center of their home municipalities. All data collection procedures were standardized. Pediatricians in training participated in all phases of data collection under the supervision of the authors of this study.p<0.25 at a time. The remaining final model included variables with p≤0.05 and those with epidemiological criteria after assessment of collinearity A binomial logistic regression model was used to assess the association of every risk factor with cases and controls in two steps. In the first, cases and controls were compared in a univariate analysis, followed by a forward multivariate analysis using every variable with In the second step, the cases/controls were stratified in two groups according to region of residence . The multivariate analysis was carried out once again, this time considering only the variables that remained in the final model as statistically significant. For every step, the fit of the multiple regression model was assessed using Hosmer & Lemeshow's statistics The association measure was the odds ratio, with confidence interval of 95% and significance level of 5% Congenital toxoplasmosis was confirmed at the age of 12 months for a total of 190 out of 146 307 screened newborns from November 2006 through May 2007. The referral outpatient clinic provided health care to 178 of these children (including one case of twins), and 12 children were followed at distance in their own municipality of residence. Only two pairs of mother/child refused to participate and therefore were excluded from the analysis. Thus, 175 mothers of infected children participated in the epidemiological interview .Toxoplasma treatment during pregnancy, and three because of reports of symptoms compatible with congenital toxoplasmosis. The final sample of 278 control mothers resulted in a case/control ratio of 1∶1.6  and number of inhabitants (p = 0.94).As expected by study design, municipalities of the cases and the controls showed no statistical difference concerning the MGSRI indicator (p<0.01). However, no statistical difference was found in relation to the risk factors reported by the mothers of the control group after categorizing the group in quartiles of the children's age.The analysis of the children's age showed that the group of cases had lower median age than the group of controls – 58 and 94 days respectively , also in Brazil, found that the only risk factor significantly associated with the toxoplasmosis prevalence was low level of education The present study showed that, in Minas Gerais, younger mothers, including adolescents, are significantly more affected by the disease, even after adjustment in the multivariate model. This association has also been found in another Brazilian study carried out in the Municipality of Goiânia, located in the midwest of the country The study also found that prenatal care might fail to guide pregnant women on how to prevent toxoplasmosis. This was true for both cases and controls, irrespective of the higher number of prenatal care appointments in the group of controls. Although quality of prenatal care was not part of this study, this finding suggests that most practitioners have neglected a great opportunity to prevent the disease.Regarding the possible sources of infection, it is well known that the importance of each varies regionally according to climate, cultural differences of hygiene and dietary habits, as well as farming practices. In Europe, the high prevalence of toxoplasmosis has been associated especially with the consumption of raw or undercooked meat infected with cysts, while in Central America and other developing countries the main factor seems to be environmental exposure to oocysts The consumption of undercooked meat was significantly associated with the infection in the univariate analysis, but did not remain as a strong variable in comparison to other risk factors included in the multivariate model.The transmission of the parasite through consumption of infected meat is conditioned to the viability of the tissue cysts. Cooking food to ≥67°C for at least 15 minutes is the safest way to eliminate the cysts, but freezing it to under −12°C overnight is also helpful, as most cysts die at cold temperatures T. gondii seropositivity in the population groups at mid or low socioeconomic levels living in Campos dos Goytacazes, a municipality in the State of Rio de Janeiro Another important risk factor that remained associated with congenital toxoplasmosis in the final model was drinking untreated and unboiled water. Several studies in other regions in Brazil have also reported such an association T. gondii oocysts in approximately 10% (21/216) of the sampled fruits and vegetables from groceries and home gardens.As for water, oocysts in soil can also contaminate fruits and vegetables. In Poland, a study, employing molecular technique, detected Some authors have reported contact with cats as a risk factor for toxoplasmosis T. gondii, this variable may indeed serve as an important environmental marker of the presence of the parasite.Another relevant aspect in our study was the association with the existence of cats in the neighborhood. Considering that the presence of cats in the neighborhood also implies the potential contamination of soil with feces containing oocysts of T. gondii infection in the rural area Toxoplasmosis is an important epidemiologic problem, especially in rural areas, where the parasite has several opportunities to spread For this reason, we opted to stratify the sample according to the domicile region. The analysis of the risk factors that remained statistically significant in the final model allowed better understanding of the transmission dynamics of the disease in each of these environments.Drinking untreated and unboiled water and living in a residence without a flush toilet were risk factors only in the group of mothers living in rural areas. Studies in Poland have stressed the importance of water as a mean of parasite dissemination in rural areas with inadequate hygiene conditions Another variable that remained statistically significant in the rural stratum was the presence of cats in the neighborhood. This constitutes a substantial problem in this population given the higher survival rates of the oocysts in non-urbanized, unpaved areas as well as the probability of oocyst dispersion by rainwater In the urban and peri-urban strata, the variables that lost significance were drinking untreated and unboiled water and living in a home without flush toilet. This finding probably reflects the more homogeneous access of the urban population to municipal water supply and sanitary infrastructure in general.This study carries some limitations, which are due not only to the case-control approach, but also to a few choices made. The most relevant limitation corresponds to the process of selecting and classifying the control group.http://tabnet.datasus.gov.br/cgi/deftohtm.exe?sinasc/cnv/nvMG.def), one can observe a significantly higher proportion of adolescent mothers and less educated mothers (less than seven years at school) among the cases (respectively 31.4% and 56.0%) than among the general population (respectively 18.7% and 39.6%). Additionally, we compared the proportion of population in Minas Gerais State served by sewage and water supply in both research periods (2007 and 2011) using available information on proxies for economic changes. They were respectively 79.4% and 79.7% for the former and 85.9% and 85.2% for the latter (http://tabnet.datasus.gov.br/cgi/idb2012/matriz.htm#socio). Although resulting in significant p values (<0.05), probably because of the sample size, we can see that those changes were rather small.Because of operational and financial constraints, we could not seek the controls in the same calendar year in which the cases were born. Although no state or national policy for control of congenital toxoplasmosis changed in study period Sampling of controls followed some criteria as a means of preserving sample representativeness, and no significant difference was found across the answers provided by random and selected controls. Another limitation was that the controls children were older than cases by the time of interview. In order to investigate a potential information bias, a sub-analysis was carried out including only the mothers (case and controls) of the children aged between 50 and 110 days. That interval was chosen considering the age median of group of controls and cases. This sub-analysis showed no overall changes in the main associations found in this study.Finally, as the children in the control group were not serologically screened for toxoplasmosis, a few cases might have been inadvertently included in the control group, reducing the association estimates. This is a remote possibility, considering the prevalence of 13 cases of congenital toxoplasmosis for every 10 000 liveborn infants in Minas Gerais. If this classification error did take place, however, it was not sufficient to hamper the identification of associations in our study.Case-control studies are classically subject to recall bias, as the mothers of the affected children may be more likely than the others to remember of factors related to their exposure In conclusion, this population-based investigation adds valuable contributions to the understanding of risk factors for congenital toxoplasmosis in Minas Gerais State. The differences between populations living in rural and urban areas regarding the main risk factors for toxoplasmosis point to the need of considering regional specificities in planning strategies to control congenital toxoplasmosis. In the urban area, most affected mothers were younger and had lower educational attainment, pointing to the potential value of focusing on primary preventing measures among these subgroups. The association between consumption of fresh meat that had not been frozen serves to warn surveillance authorities to implement careful inspection of the meat available for human consumption. In the rural area, where risk factors related to the ingestion of oocysts and poor sanitation predominated, priority should be given to the public measures aimed at improving sanitation.


KCNJ11) variation in susceptibility to type 2 diabetes (T2D), we performed a systematic meta-analysis to investigate the association between the KCNJ11 E23K polymorphism (rs5219) and the T2D in different genetic models. Databases including PubMed, Medline, EMBASE, and ISI Web of Science were searched to identify relevant studies. A total of 48 published studies involving 56,349 T2D cases, 81,800 controls, and 483 family trios were included in this meta-analysis. Overall, the E23K polymorphism was significantly associated with increased T2D risk with per-allele odds ratio (OR) of 1.12 . The summary OR for T2D was 1.09 , and 1.26 , for heterozygous and homozygous, respectively. Similar results were also detected under dominant and recessive genetic models. When stratified by ethnicity, significantly increased risks were found for the polymorphism in Caucasians and East Asians. However, no such associations were detected among Indian and other ethnic populations. Significant associations were also observed in the stratified analyses according to different mean BMI of cases and sample size. Although significant between study heterogeneity was identified, meta-regression analysis suggested that the BMI of controls significantly correlated with the magnitude of the genetic effect. The current meta-analysis demonstrated that a modest but statistically significant effect of the 23K allele of rs5219 polymorphism in susceptibility to T2D. But the contribution of its genetic variants to the epidemic of T2D in Indian and other ethnic populations appears to be relatively low.To clarify the role of potassium inwardly-rectifying-channel, subfamily-J, member 11 ( By coupling cell metabolism with membrane potential, adenosine triphosphate-sensitive potassium channel (KATP) play a central role in regulation of insulin secretion in pancreatic-β cells. 2-terminal tail of Kir6.2 was identified KCNJ11 on T2D risk.As a candidate gene for T2D in humans, a nonsynonymous E23K variant (rs5219) which results from a G → A transition in codon 23 in the NHKCNJ11 gene were retrieved. Search keywords combinations were “potassium inwardly-rectifying-channel, subfamily-J, member 11”, “KCNJ11”, “Kir6.2”, “type 2 diabetes”, “type 2 diabetes mellitus”, “T2D”, “T2DM”, “non-insulin-dependent diabetes mellitus”, “NIDDM”, “polymorphism” or “variation”. The titles and abstracts were read to determine their relevance, and potentially relevant studies were retained for further evaluation. For retrieved articles, the full texts were carefully read to determine whether they meet the purpose of the present meta-analysis. Furthermore, the references of these studies were checked to identify other relevant publications.To identify eligible literatures, we conducted a computer-based search of PubMed, Medline, EMBASE and ISI Web of Science databases without language restrictions. Studies published before the end of Mar. 2013 on T2D and the E23K polymorphism in the KCNJ11 E23K polymorphism with T2D risk (2) being case-control or cohort studies (3) diagnosis of T2D patient was confirmed pathologically and (4) providing sufficient data for calculation of odds ratio (OR) with its 95% confidence interval (95% CI) and P-value. The major reasons for exclusion were (1) case-only studies (2) overlapping data (3) review papers. If more than two studies reported the same sample, only the study providing more information or latest published was selected.Eligible studies should meet following criteria: (1) focusing on the association of the The following information was carefully extracted from all eligible publications: the first author, year of publication, country of origin, ethnicity of subjects, study design, sample size, sex distribution among cases and controls, mean age and body mass index (BMI) of cases and controls, source of control, Hardy–Weinberg equilibrium (HWE) status in controls, genotyping method, number of genotypes in cases and controls. Two authors independently assessed the articles for compliance with the inclusion criteria, and disagreement was followed by discussion until consensus was reached.KCNJ11 E23K polymorphism with T2D was evaluated by calculating a pooled OR and 95% CI for allele contrast , heterozygous (KE vs. EE) and homozygote (KK vs. EE). Then, we examined the association between the polymorphisms and T2D risk using dominant and recessive genetic models. The standard Q-statistic test was performed to evaluate whether the variation between studies was due to heterogeneity or due to chance The association of the We calculated the sample size required for 80% power with the summary OR estimated from each ethnic populations, assuming an equal number of cases and controls, risk allele frequency (RAF) in controls estimated from different ethnicity. Furthermore, population attributable risk (PAR) was calculated to get a comprehensive view of the impact of the E23K variant on T2D at population level. PAR was calculated by the following formula: (OR-1)/OR * risk allele frequency Egger's test and funnel plots were used to assess small studies effects KCNJ11 E23K polymorphism The literature search yielded 159 studies using keywords listed above. KCNJ11 E23K polymorphism and T2D were detected when all the eligible studies were pooled into the meta-analysis  and homozygous  when compared with wild type homozygous. Similar results still maintained using dominant and recessive genetic models (P<10−5) and of 1.12  respectively. Significantly increased risks were also found for heterozygous and homozygous .Overall, significant associations between analysis . The ove P<10−5; . Signific models . When stmozygous . Howevermozygous . For twoP<10−5). Hence, meta-regression was further conducted to investigate the source of heterogeneity. In meta-regression analysis, ethnicity (P = 0.79), sample size (P = 0.61), mean age (P = 0.36) of cases and controls (P = 0.61), gender distribution in cases (P = 0.96) and controls (P = 0.30) did not explain a large part of the heterogeneity among the individual study. By contrast, mean BMI (P = 0.03) explained about 11% of the heterogeneity.Significant heterogeneity was found among the 46 included studies .The results of sensitivity analysis confirmed the significant associations of the tatively . The EggLimited statistical power of relative small sample size is a common problem in genetic association for individual T2D studies. Therefore, sufficient sample power is necessary in deciphering genetic architecture of T2D, but it is sometimes very difficult for a single study to collect enough amounts of data to reach a reliable conclusion. By pooling of data from individual association studies, meta-analysis is an effective approach of increasing the sample size under investigation, thus enhancing the statistical power for the estimation of genetic effects.KCNJ11 is a risk factor for developing T2D. In the subgroup analyses by ethnicity, we found the rs5219 polymorphism was associated with T2D among East Asians and Caucasians, but not Indians or other ethnic populations. Of note, different ethnic populations were pooled in the other ethnic group and only a few studies were available in the subgroup, so the result must be interpreted with caution. There are several other possible reasons which may account for such differences. First, T2D is a complex disease and different genetic backgrounds may cause the discrepancy since the distributions of the risk-association alleles in KCNJ11 were different between various ethnicities. The K allele frequency of the rs5219 polymorphism was ∼36%, ∼40%, and ∼34%, among East Asians, Indians and Caucasians populations, respectively. Such result could also be due different linkage disequilibrium (LD) pattern of the polymorphism and nearby causal variant among different ethnic populations. Moreover, inter-individual difference like age, sex, dietary intake of nutrients, in addition to phenotype heterogeneity, such as years from onset and severity of the disease may also explain the discrepancy. Furthermore, study design and/or small sample size or some environmental factors may also affect the results. Therefore, more studies are needed to further validate the effect of the polymorphism on T2D risk among difference ethnic populations.Our results indicated that the rs5219 polymorphism of KCNJ11 gene has attracted considerable attention as a promising candidate for T2D based on its position and its function as a key factor in the regulation of glucose-induced insulin secretion, since normoglycemic lysine carriers are shown to consistently display a defect in insulin secretion KCNJ11 E23K variant was found to be associated with glucose intolerance and conversion from impaired glucose tolerance to T2D among Caucasians in vitro properties of KATP channel via increasing the threshold of ATP concentration for insulin secretion The The distribution of the E23K variant in controls across various studies showed global variation , suggestKCNJ11 as a risk region in T2D susceptibility which was in line with the results the present meta-analysis.Power analysis revealed a hint in design for future association studies. So far, most association studies have included at the most several hundred subjects and results from these studies should be treated with caution as for limited statistic power to reach a reliable conclusion. According to our power analysis, powerful association studies on the E23K polymorphism and T2D risk may need several thousand individuals. Compared with candidate gene approach, genome wide association study (GWAS) with large sample size and unbiased to genomic structure is a powerful approach in susceptible gene identification for T2D. Recently, Tsai et al. KCNJ11 and genetic susceptibility to T2D.In comparison with the previous published meta-analysis Heterogeneity among pooled studies is a frequently encountered issue in meta-analyses. Of note, our meta-analyses joined population bases association studies and family based association studies, which could enhance the heterogeneity. Hence, we performed subgroup analysis and meta-regression to identify potential source of between-study heterogeneity. However, they revealed that only BMI of controls could explain a small part of significant heterogeneity between studies. There may be a number of possible underlying reasons. Firstly, results from case–control studies may differ because of ethnic diversity  and geographic variation. Secondly, variations in methods of sample ascertainment and diagnosis may also contribute to such inconsistence. Thirdly, environmental factors, such as alcohol drinking, smoking behavior, obesity might also result in variability between studies.KCNJ11 and T2D. Our results suggest a modest but statistically significant effect of the 23K allele of rs5219 in susceptibility to T2D, particularly in East Asians and Caucasians. More work will be required for future association studies, especially those which are properly powered, effectively control for confounding factors, employing family-based design. Moreover, gene–environment and gene–gene interactions should also be taking into consideration for future studies.To conclude, this may be the most comprehensive meta-analysis of Checklist S1(DOC)Click here for additional data file.Figure S1Flow chart of literature search for studies examining KCNJ11 rs5219 polymorphism and risk of T2D.(TIF)Click here for additional data file.Figure S2Result of sensitivity analyses.(TIF)Click here for additional data file.Figure S3Begg's funnel plot of rs5219 polymorphism and T2D risk.(TIF)Click here for additional data file.Table S1Results of meta-analysis for KCNJ11 E23K polymorphism and T2D risk using dominant and recessive genetic models.(DOCX)Click here for additional data file.


Background and Objectives. Migraine is a multifactorial common neurovascular disease with a polygenic inheritance. Inflammation plays an important part in migraine pathophysiology. C-C chemokine receptor 2 (CCR2) is an important chemokine for monocyte aggregation and transendothelial monocyte migration. The aim of our study was to investigate the association of migraine with CCR2V64Il polymorphism in the Iranian population. Methods. We assessed 103 patients with newly diagnosed migraine and 100 healthy subjects. Genomic DNA samples were extracted from peripheral blood and genotypes of CCR2V64Il gene polymorphism were determined. For measuring the severity of headache, every patient filled out the MIGSEV questionnaire. Results. There were no significant differences in the distribution of both 64Il allele and heterozygote (GA) genotype of CCR2 gene polymorphism  between case and control groups. There was no significant difference of alleles frequency between three grades of MIGSEV (P = 0.922). Conclusions. In conclusion our results revealed no association between CCR2V64Il polymorphism and susceptibility to migraine and also headache severity in the Iranian population. Migraine is a common neurovascular disease characterized by recurrent pulsating and unilateral headache attacks, associated with autonomic symptom such as nausea, vomiting, phonophobia, and photophobia. Some patients have transient neurologic attacks known as “aura” . The preMigraine is a multifactorial disorder with a polygenic inheritance. Different mechanisms can be related to migraine such as altered sensory input, increased sensitivity of cortex that induces aura, neurogenic inflammation, and hypersensitivity of nociceptors, and sensory neurons that response to pain, in the brain stem .Neurogenic inflammation implicates activation of inflammatory agents and sensitization of nociceptor that innervate the meninge .Chemokines and chemokine receptors play a crucial role in inflammatory processes. Chemokines are proinflammatory cytokines that attract leukocytes and promote accumulation of these cells at the source of chemokine production . ChemokiCCR2 gene is localized on the chemokine receptor gene cluster on chromosome 3. CCR2A and CCR2B are two alternatively spliced transcript variants. This gene has three exons and spans approximately 8 kb. Most of the studies focus on the role of CCR2 in inflammatory and autoimmune diseases. CCR2 plays a major role in vascular inflammation and developing atherosclerotic plaques , 8 and aIn recent years most of the studies have focused on the Val64Ile variant of CCR2 , 10. CCRDifferent studies have investigated the association of CCR2V64Il with several inflammatory diseases and have reported controversial results. CCR2V64Il is associated with coronary artery disease (CAD)  and alsoWe conducted a case-control study enrolling patients that were recently diagnosed with migraine without aura based on the International Headache Society (IHS) criteria  and alsoEach migraine patient completed a MIGSEV questionnaire as a valid scale for assessing headache severity. The MIGSEV scale, developed by EL Hasnaoui et al. in 2003, is a simple severity scale with four items including intensity of pain, disability in daily activity, tolerability, and nausea that categorizes patients in three groups of intensity: mild, moderate, and severe . This inTwo mL of venous blood was collected from each participant. Genomic DNA samples were extracted from peripheral whole blood using the AccuPrep Genomic DNA Extraction kit  according to the manufacturer's protocol.NCBI data bank and primers were designed by Beacon Designer 7.91 to flank the coding regions  and synthesized by TIB MOLBIOL, Germany. The forward primer was 5′-ACGGTGCTCCCTGTCATAA-3′ and reverse primer was 5′-CATTCCCAAAGACCCACTCATT-3′.The SNP rs1799864 was identified by the Genotyping was done by high-resolution melt (HRM) assay using a Rotor-Gene 6000 instrument .μL of final volume using the Type-it HRM kit (Qiagen), HRM PCR buffer, HotStarTaqPlus DNA Polymerase, nucleotides and EvaGreen dye, and 30 ng DNA.PCR reactions were carried out in duplicate in 20 The PCR program consisted of an initial denaturation-activation step at 95°C for 5 min, followed by a 40-cycle program .Curves for each duplicate were checked on the shape and peak height to meet reproducibility.Normalized and temperature-shifted melting curves from HRM, suggestive of single nucleotide polymorphisms (SNP), were distinguished and the samples were subjected to direct sequencing.t-test was used for quantitative variables between two groups. Relation between polymorphism (homozygous and heterozygous) and different categorized variables,  was established using Chi-square test and calculation of odds ratio (CI = 95%). The significant level was considered as P < 0.05.The Hardy-Weinberg equilibrium (HWE) was tested by a goodness-of-fit Chi-square test to compare the observed genotype frequencies to the expected frequencies among controls. We analyzed our data with SPSS software . An independent DNA samples from 103 subjects with migraine and 100 healthy subjects were analyzed for CCR2 gene polymorphism. In the case and control groups 82.6% and 78% of participants were female, respectively, and the difference was not statistically significant. Also there were no significant differences in the mean ages (34.08 ± 1.01 versus 34.77 ± 1.06 years), education level (40% versus 36% without academic degrees), and residency (51.5% versus 57.6% urban) between case and control groups, respectively. Subjects' characteristics of the case group are reported in P = 0.396) (P = 0.388)  and 49 (12.1%), respectively. The A allele distribution (Minor Allele Frequency) was not significantly different between migraine patients and control subjects  . The fre= 0.388) .P = 0.162 and 0.321, resp.). Further analysis in the migraine subjects showed that the distribution of CCR2 gene polymorphism was not associated with the presence of family history of migraine (P = 0.944). We classified all migraine patients into three groups according to the MIGSEV scale. There was no significant difference of alleles frequency between three grades of MIGSEV (P = 0.922). Also comparison of the frequency of headache per month between the homozygote and heterozygote patients was not significantly different .The comparison of allele frequencies, between males and females in the case and also control groups, showed that there is no significant difference in the alleles distribution according to the sex  which is an important factor in regulating neural plasticity and inflammatory responses is a target of CCR2 signaling. This fact indicates that CCL2-CCR2 may play a role in neuroinflammation and chronic pain . Also AbPrevious studies indicated a relation between CCR2V64Il, a variant of CCR2, and some inflammatory and autoimmune diseases such as coronary artery disease, diabetes mellitus, progression of HIV, chronic renal failure, and trigeminal neuralgia , 27, 28.There are only two studies about the relation between migraine and chemokine receptor polymorphisms. Combadière et al. evaluated the relation between headache and CX3CR1 polymorphism. This study showed that CX3CR1 polymorphism had no association with migraine but may play a role in recurrent headaches. This study has some limitations such as misclassification in assessing the type of headache because of recall bias related to elderly participants of this study .Another study was done on the relation between migraine and CCR2V64IL. This study evaluated 77 polymorphisms in a large number of patients with migraine and in contrary to our study showed the correlation of CCR2V64Il and migraine but the association did not remain significant after adjustment for multiple testing. However, this study was conducted only on white females more than 45 years old .In conclusion our result revealed no association between CCR2V64Il polymorphism and susceptibility to migraine without aura and also headache severity in the Iranian population. However, we enrolled newly diagnosed patients by neurologist without the history of receiving any specific medications for migraine which resulted in a relatively small sample size and thus a limitation to our study. The small sample size reduced the power of our study. Especially, in analysis of the association between allele frequency and the three different groups of MIGSEV grades. Therefore this is a preliminary conclusion. Further studies with larger sample sizes from a more diverse ethnic population are needed to confirm these findings.


Hepatocarcinogenesis is a complex process that may be influenced by many factors, including polymorphism in microsomal epoxide hydrolase (mEH). Previous work suggests an association between the Tyr113His and His139Arg mEH polymorphisms and susceptibility to hepatocellular carcinoma (HCC), but the results have been inconsistent.PubMed, EMBASE, Google Scholar and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. A meta-analysis was performed to examine the association between Tyr113His and His139Arg mEH polymorphism and susceptibility to HCC. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated.Eleven studies were included in the meta-analysis, involving 1,696 HCC cases and 3,600 controls. The 113His- mEH allele was significantly associated with increased risk of HCC based on allelic contrast , homozygote comparison  and a recessive genetic model , while individuals carrying the Arg139Arg mEH genotype had no association with increased or decreased risk of HCC.The 113His- allele polymorphism in mEH may be a risk factor for hepatocarcinogenesis, while the mEH 139Arg- allele may not be a risk or protective factor. There is substantial evidence that mEH polymorphisms interact synergistically with other genes and the environment to modulate risk of HCC. Further large and well-designed studies are needed to confirm these conclusions. As the most frequent primary cancer of the liver, hepatocellular carcinoma (HCC) is a significant cause of cancer morbidity and mortality worldwide, and treatment options are limited. The estimated incidence of new HCC cases each year is more than 0.5 million HCC pathogenesis remains incompletely understood. It is known to involve chronic inflammation, hepatocyte hyperplasia and ultimately malignant transformation The liver has a complex detoxification system. In this complex system, several enzymes participate in the metabolism of both exogenous and endogenous metabolites produced by oxidative stress in chronic infections, such as HBV and HCV infections. The genes encoding these enzymes are polymorphic, and the variant sequences lead to enzymes with altered activity. Any genetic defect in these xenobiotic-metabolizing enzymes may lead to accumulation of viral integration products and xenobiotics. This should increase the risk of liver diseases, such as cirrhosis and HCC.Microsomal epoxide hydrolase (mEH) is one of these enzymes. As a phase II metabolic enzyme, mEH is involved in the hydrolysis of various epoxides and deleterious chemical epoxide intermediates generated by phase I oxidation reactions The gene encoding mEH consists of nine exons and eight introns. Two relatively frequent single nucleotide polymorphisms occur in the coding region: Tyr113His in exon 3 and His139Arg in exon 4. These polymorphisms modify protein stability: Tyr113His decreases enzyme activity, while His139Arg increases it In 1995, Mcglynn and coworkers mEH; HYL1; EPHX; microsomal epoxide hydrolase; these four terms in combination with polymorphism, variation, genotype, genetic and mutation; and all of the above terms in combination with hepatocellular carcinoma, HCC, liver cancer, liver tumor, liver neoplasms and hepatic tumor. The references of each article identified were also manually searched to identify additional relevant publications.All case-control studies of mEH polymorphism and HCC risk published up to August 31, 2012 were identified through systematic searches in PubMed, EMBASE, Google Scholar and the Chinese National Knowledge Infrastructure (CNKI) databases using English and Chinese. The search terms used were: A study was included in the meta-analysis if it satisfied the following criteria: (a) it assessed the association between HCC and the Tyr113His and His139Arg mEH gene polymorphisms, (b) it used a case-control design, and (c) it provided sufficient published data for estimating an odds ratio (OR) with a 95% confidence interval (95% CI). In the case of multiple studies based on the same population, we selected the study with the largest number of participants.Literature searches and identification of eligible articles based on the inclusion criteria were carried out independently by two authors (JHZ and BDX). These authors independently extracted the following data: first author’s name, year of publication, ethnicity or country, source of controls , exon number, numbers and genotypes of cases and controls, and Hardy-Weinberg equilibrium (HWE) of controls. Discrepancies were resolved by consensus.The unadjusted OR with 95% CI was used to assess the strength of the association between the Tyr113His and His139Arg mEH polymorphisms and HCC risk based on the genotype frequencies in cases and controls. Subgroup analysis stratified by ethnicity was performed. Ethnicity was categorized as Chinese and mixed. The meta-analysis examined the association of different genotypes at Tyr113His mEH with HCC risk by comparing the 113 Tyr allele  with the 113 His allele , the homozygous comparison (His113His vs. Tyr113Tyr), and recessive and dominant genetic model. The same approach was used to examine the association of His139Arg mEH and HCC risk.All statistical tests for this meta-analysis were performed using RevMan 5.0 and Stata 11.0 softwares. Fixed-effect and random-effect models were used to calculate a pooled OR. The statistical significance of the pooled OR was determined using the Z-test, and P<0.05 was considered statistically significant. The assumption of heterogeneity was evaluated by applying a chi square-based Q-test among the studies. P>0.10 for the Q-test indicates a lack of statistical heterogeneity, suggesting the variability in effect sizes is larger than that expected from chance alone A total of 223 potentially relevant publications up to August 31, 2012 were systematically identified through PubMed, EMBASE, Google Scholar and CNKI. After screening the title and/or abstracts, 174 (78%) were excluded because they did not satisfy the inclusion criteria. An additional 36 publications were excluded because they did not examine Tyr113His and His139Arg mEH polymorphisms or they were review articles. Three publications We established a database according to the information extracted from each article. Detailed characteristics of the 11 studies are listed in All 11 studies described the Tyr113His polymorphism; however, only 6 studies P = 0.003, I2 = 72%) and the dominant genetic model comparison . However, no heterogeneity was found between studies for the homozygote comparison and recessive genetic model comparison (I2 = 0%)  .P = 0.02; I2 = 83%)  and the recessive genetic model . Association of the mEH with HCC risk was not observed in the total population using the dominant genetic model .Calculation of overall OR in the total population using the random-effect model showed that the 113His- allele was strongly associated with increased risk of HCC based on allelic contrast  , homozygP = 0.30; I2 = 72%), homozygote comparison , dominant genetic model comparison  or recessive genetic model comparison . Because the populations in the six studies reporting the His139Arg polymorphism were from non-overlapping ethnicities, we did not conduct subgroup analysis by ethnicity.For the His139Arg mEH polymorphism and HCC risk, no statistically significant effect was observed based on allelic contrast . However, this association was not observed in the dominant genetic model .After stratification for ethnicity, we observed that in the Chinese population, the 113His- allele, homozygote variant and recessive genetic model were significantly associated with increased risk of HCC . Similarly, the dominant Tyr113His genotype was not associated with HCC risk.Analysis of the mixed population in five studies P = 0.08). In the recessive genetic model contrast, the results were not altered after excluding studies one by one. However, in the dominant genetic model contrast, the result was statistically significant after excluding the study by Kiran et al. P = 0.02). Therefore, the results should be interpreted with caution. For the His139Arg mEH polymorphism and HCC risk, the results were not altered based on allelic contrast, dominant or recessive genetic model comparison.In the allele contrast, the result was altered after excluding the study by Mcglynn and coworkers Harbord’s modified test was prepared for the 11 studies HCC involves complex, multistep and heterogeneous malignant tumorigenesis. The pathogenesis of HCC involves host and environmental factors, as well as modulation of molecular signaling pathways implicated in malignant transformation of hepatocytes and tumor progression Some studies reported an association between the Tyr113His polymorphism and HCC risk, while others found no such association. The most likely reason for the inconsistencies among these studies is that they are single case-control studies with small sample sizes. To help resolve these conflicting results using a larger sample size, we conducted meta-analysis of published studies. Our results for the total population suggest an increased HCC risk for subjects carrying the Tyr113His mEH genotype. Our approach also allowed us to look for potential ethnic differences in the association. Analysis of ethnic subgroups showed that in the Chinese population, the 113His- allele was strongly associated with increased risk of HCC based on allelic contrast, homozygote comparison and the recessive genetic model. However, no association was observed for other populations . Six studies The clinical significance of mEH polymorphisms extends beyond HCC. Polymorphisms in this gene have been reported to be susceptibility factors for several human diseases, such as type 2 diabetes mellitus Several studies in our meta-analysis indicate that mEH polymorphisms can interact with environmental factors to module HCC risk  [10], [1Our meta-analysis also revealed gene-gene interactions between the mEH polymorphisms and genotypes associated with elevated risk of AFB toxicity. Individuals with a greater number of suspected AFB-related high-risk genotypes  faced a greater risk of HCC than those without any high-risk genotypes, especially among individuals with higher AFB exposure The 113His codon variant is relatively common in the mEH gene. However, the allele frequency differs significantly among ethnicities. Kiyohara et al. Our data revealed that His113His mEH is a risk factor for hepatocarcinogenesis, but the complete picture is more complex. Both China and Gambia have among the highest incidences of HCC in the world, as well as a high prevalence of HBV infection and dietary exposure to AFB, which are the two main risk factors for HCC Of the total number of 5,296 subjects considered in the meta-analysis, at least 2,000 (37.8%) had one or more of the following: alcoholic liver disease, HBV or HCV infection, and cirrhosis. Since the studies in our meta-analysis often did not report detailed statistics on the proportion of HCC or control subjects with these background conditions, we could not perform subgroup analysis to separate the contribution of mEH polymorphism from that of possible confounders like HBV or HCV infection. Some other limitations of this meta-analysis also should be considered when interpreting the results. Bias may result from our exclusion of unpublished data, as well as papers published in languages other than English and Chinese In conclusion, this meta-analysis suggests that the His113His mEH polymorphism may be associated with increased risk of HCC, especially in the Chinese population. Moreover, the mEH gene is involved in synergistic gene-gene and gene-environment interactions. These results suggest that sequence variation in exon 3 of the mEH gene may play an important role in the occurrence of HCC. In contrast, no association was observed between mEH gene variation in exon 4 and the risk of HCC. However, since this meta-analysis included few studies from non-Asian populations, large, well-designed studies in Caucasian and African populations are warranted to re-evaluate these associations.This meta-analysis is guided by the PRISMA statement (Checklist S1).Table S1Overall and stratified meta-analyses of the association between mEH polymorphism His139Arg and risk of hepatocellular carcinoma.(DOC)Click here for additional data file.


Nitric oxide (NO) has numerous functions in the kidney, including control of renal and glomerular hemodynamics, by interfering at multiple pathological and physiologically critical steps of nephron function. Endothelial NOS (eNOS) gene has been considered a potential candidate gene to diabetic nephropathy (DN) susceptibility. Endothelial nitric oxide synthase gene (eNOS-3) polymorphisms have been associated with DN, however some studies do not confirm this association. The analyzed polymorphisms were 4b/4a, T-786C, and G986T.st, 2012 were identified by searching electronic databases. Hardy-Weinberg equilibrium assessment was performed. Gene-disease association was measured using odds ratio estimation based on the following genetic contrast/models: (1) allele contrast; (2) additive model; (3) recessive model; (4) dominant model and (4) co-dominant model.The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement was used in this report. Case–control studies that had diabetic patients with DN as cases and diabetic patients without nephropathy as controls, as well as that evaluated at least one of the three polymorphisms of interest were considered eligible. All studies published up until December 31Twenty-two studies were eligible for meta-analysis . Considering 4b/a polymorphism, an association with DN was observed for all genetic models: allele contrast ; additive ; recessive ; dominant , with the exception for co-dominance model. As well, T-786C polymorphism showed association with all models, with exception for co-dominance model: allele contrast , additive , recessive , and dominant . For the G894T polymorphism, an association with DN was observed in allelic contrast  and co-dominance models .In the present study, there was association of DN with eNOS 4b/a and T-786C polymorphism, which held in all genetic models tested, except for co-dominance model. G894T polymorphism was associated with DN only in allele contrast and in co-dominance model. This data suggested that the eNOS gene could play a role in the development of DN. Nitric oxide (NO) is a short-lived gaseous lipophilic molecule produced in almost all tissues and organs,2. It isNO has numerous functions in the kidney, including control of renal and glomerular hemodynamics, by interfering at multiple pathological and physiologically critical steps of nephron function. NO dilates both the afferent and the efferent arteriole, augmenting the glomerular filtration rate (GFR) and influencing renal sodium handling. NO alsoeNOS gene has been considered a potential candidate gene to diabetic nephropathy (DN) susceptibility. Since 1998, several polymorphisms of the eNOS gene have been identified, and their association with various diseases has been explored. Three polymorphisms have been the subject of research in relation to DN, however the results are highly variable. The polymorphisms potentially associated with DN are a 27-bp repeat in intron 4 (VNTR), the T-786C single nucleotide polymorphism (SNP) in the promoter region (rs2070744), and G894T missense mutation in exon 7 (rs1799983)[However, the potential association of eNOS gene variants with the induction and progression of DN remains controversial. Some authors found a higher frequency of eNOS polymorphisms in patients with end-stage renal disease (ESRD) and DN-17, but The objective of the present study was to evaluate if eNOS gene polymorphisms are associated with DN through a systematic review of the literature and a meta-analysis.Three-hundred and nine studies were identified, and 281 were excluded based on review of titles and abstracts . Twenty-eight articles were eligible and had the full text evaluated. Six studies were excluded due to lack of information regarding genotypic distribution. A total of 22 studies fulfilled the eligible criteria and were included for the meta-analysis  was assessed using exact test and P-value < 0.05 were considered significant. Only 4 studies  with controls were not in HWE ; additive : 1.18-1.97, PQ <0.01, I2 = 62%); recessive : 1.16-1.93, PQ <0.01, I2 = 64%); and dominant : 1.01-1.23, PQ = 0.01, I2 = 49%). Similarly, for the T-786C polymorphism the association with DN was found with all models, with exception for co-dominance model: allele contrast : 1.07-1.39, PQ = 0.59, I2 = 0%), additive : 1.18-1.97, PQ < 0.01, I2 = 62%), recessive : 1.16-1.93, PQ <0.01, I2 = 64%) and dominant : 1.01-1.23, PQ <0.01, I2 = 49%). The G894T polymorphism showed association with DN in allelic contrast : 1.03-1.25, PQ <0.01, I2 = 75%) and co-dominance model : 1.04-1.37, PQ = 0.01, I2 = 60%) : 1.05-1.25, PPublication bias was observed for the majority of the polymorphisms evaluated and are presented as a funnel plot for 4b/a polymorphsism Figure . In ordeSince some studies included only subjects of specific ethnicities or with type 1 or type 2 DM, we performed a sensitive analysis stratifying the studies according to these characteristics. Considering 4b/a polymorphism, there was an association in White and East Asian populations in allele contrast, additive and recessive models; only for Whites in the dominant model; and none for the co-dominant model. For T-786C variant, no association was shown for Whites in allele contrast analysis or in any other genetic model, but in African populations the polymorphism was associated with DN in allele contrast, dominance and co-dominance models. Considering G894T polymorphism, in African populations the association was observed for all genetic models, with the exception of co-dominance model. There were insufficient studies to perform a meta-analysis for G894T in South Asians and West Asians.According to the type of diabetes mellitus (DM), for 4b/a polymorphism an association was observed in additive and recessive models for both type 1 and type 2 diabetes, and only for type 1 in allele contrast and dominant models. There was no association with any type of DM in co-dominant model for 4b/a variant. For T-786C, no association in any genetic model was found in type 2 diabetes. There was insufficient data for this analysis in type 1 DM. Likewise, for G894T variant there was an association only in the allele contrast model with type 2 diabetes Table .We compared the ORs of our meta-analysis with the results from a previous meta-analysis that used non-diabetic patients as controls. The resIn the present study, the most robust association of DN was with eNOS 4b/a and T-786C polymorphism that held in all genetic models tested, except for co-dominance model. G894T polymorphism was associated with DN only in allele contrast and in co-dominance model. 4b/a polymorphism association with DN was confirmed in all ethnic groups evaluated and for all types of diabetes. The subgroup analysis of the T-786C variant should be viewed with caution, since it was limited due to the small number of studies.Analyzing genetic model is important, considering the difference between them. Each individual genotype is formed by two alleles (for example G and T for G984T polymorphism), and the risk of every genotype depends on the number of variant allele copies carried, where one of which is thought to be associated with a disease , association studies will collect information on the numbers of diseased and disease-free subjects with each of the three genotypes . So we used the allele contrast, which compares the number of alleles G with the number of alleles G; the additive model, which contrasts extreme homozygotes, comparing the genotype GG with the genotype TT; in recessive model two copies of T allele are essential to modify the risk, combining the GG and GT genotypes and comparing with TT; the dominant model, which heterozygous GT and homozygous TT genotypes have the similar risk as a single copy of T is sufficient to alter the risk, then compares GG with combined GT and TT genotypes; and the codominance model, commonly used genetic model, where each genotype gives a diverse and non additive risk. which combines the GG and TT genotypes and compares with GT. So OR in each particular genetic model gives us different interpretations about the risk of the polymorphisms.These results are original and help to understand the role of these polymorphisms in the development of DN. However, it was not possible to exclude a publication bias of negative studies. Therefore, the exact effect could be smaller. As discussed before, other explanations, besides classic risk factors, are needed for understanding the progression of a diabetic patient from normoalbuminuria to macroalbuminuria, and a polymorphism identification of a specific gene would propitiate the development of a new therapy aimed directly to it.In contrast to a recent meta-analysis performed by Zintzaras et al., which aIn this sense, we considered that the optimal control group when studding a DM complication is a diabetic patient without the complication and with disease duration long enough the permit a genetic predisposition to become clinically detected in the presence of hyperglycemia. Moreover, the disease duration must be comparable between cases and controls. Most included studies fulfilled the two pre-requisitions. As can be seen in Table Despite the different control used by Zintzaras, they found 92 articles, being 20 included for meta-analysis; that provided 1942/1461 cases/controls for G894T, 2663/2232; cases/controls for 4b/a, and 857/845 cases/controls for T-786C. That was similar to ours that had 22 studies included, but provided about one third more cases/controls. The OR observed in their analyzes showed significance in allelic contrast model for G894 polymorphism, recessive and additive model for 4b/a polymorphism, and allelic contrast model for T-786C, all observed in our study; but our analyze showed association in more genetic models than that, like codominant model for G894T; allele contrast and dominant model for 4b/a; recessive, dominant and additive model for T786C. Furthermore, we compared our ORs with those reported by Zintzaras et al. and no statistical differences were found. With that said, our study reinforce the findings from Zintzaras.DN development predisposition has not been fully explained, since glycemic control and environmental factors, as well as traditional risk factors, do not accurately predict the occurrence of this diabetic complication in all patients. With this in mind, studies have been trying to resolve this question using genetic approaches. Many candidate genes have been explored in this context, and eNOS polymorphisms have been implicated in the susceptibility to glomerular disease, by mechanisms yet unknown. HoweverThe present paper has some limitations. The inclusion of studies evaluating patients with DM in several stages of DN, ranging from microalbuminuria to chronic renal insufficiency in kidney replacement therapy, could bias the results due to clinical heterogeneity of cases. Some studies did not present the data separated by DN stages. Furthermore, inclusion criteria in the reviewed studies utilized different methods and cutoffs to define microalbuminuria or macroalbuminuria. Although all clinically validated, these aIn conclusion, this study shows an association between DN and polymorphisms in eNOS gene. This effect is very consistent for the 4b and T-786 polymorphism.The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement was used in this report,37.st, 2012 were identified by searching electronic databases: Medline (1966-Present), EMBASE (1980-Present), LILACS and Cochrane Library.Case–control studies that had diabetic patients with DN as cases and diabetic patients without nephropathy as controls, as well as that evaluated at least one of the three polymorphisms of interest  were considered eligible. Only studies in humans and using validated genotyping methods were considered. No publication language, publication date, or publication status restrictions were imposed. All studies published up until December 31Abstracts presented at scientific events held by: The American Diabetes Association (ADA); The European Association for the Study of Diabetes (EASD); The National Kidney Association (NKA); and The American Society of Nephrology (ASN) were searched over the last seven years. The authors were contacted for more details in the case of abstracts with missing information.The following index terms were used:  AND  AND .Eligibility assessment was made by title and abstracts review and in doubtful cases by full article review. This was performed independently in a standardized manner by two investigators (BSD and CBL). Disagreements between reviewers were resolved by consensus.Two investigators extracted the data, one independent to another (BSD and LCFP). Disagreements were resolved by a third author (LHC). For articles with missing information, (n = 3) the authors were contacted for further information, but none responded. In the case of duplicate publications, the first manuscript published was included in the analysis. Information was extracted from each individual study based on: (1) characteristics of study participants , (2) cashttp://ihg.gsf.de/cgi-bin/hw/hwa1.pl).To ascertain the validity of each eligible case–control study, two investigators (BSD and LCFP) worked independently during the initial search and after worked together to determine the adequacy of studies selection. It was assessed if the same exclusion criteria for cases and controls were used; if cases were easily differentiated from controls; if analysis of studied polymorphisms were conducted in a standard, valid, and reliable way, if major biases were identified and considered in design and analysis; and how good the study was to minimize the risks of bias or confusion. Hardy-Weinberg equilibrium assessment among the control group within each polymorphism in all studies was checked by exact test using an online HWE calculator , and random models when heterogeneity was observed. Multiple comparisons were not made because meta-analysis of genetic association studies is considered an exploratory study, without a prespecified key hypothesis[Gene-disease association was measured using odds ratio estimation based on the following genetic contrast/models: (1) allele contrast; (2) additive model; (3) recessive model; (4) dominant model and (4) co-dominant model,40. Hetepothesis,42.The risk of publication bias was evaluated using funnel plot graphics.Sensibility tests were made concerning to ethnia and type of diabetes.http://www.stata.com).Data were analyzed using Stata/SE 11.2 (We compared the ORs of our meta-analysis with the results from a previous one that useADA: The American Diabetes Association; ASN: The American Society of Nephrology; DM: Diabetes mellitus; DN: Diabetic nephropathy; EASD: The European Association for the Study of Diabetes; eNOS: Endothelial nitric oxide synthase; ESRD: End stage renal disease; GFR: Glomerular filtration rate; iNOS: Inducible nitric oxide synthase; NKA: The National Kidney Association; nNOS: Neuronal nitric oxide synthase; NO: Nitric oxide; NOS: Nitric oxide synthase; PRISMA: The preferred reporting items for systematic reviews and meta-analysis; SNP: Single nucleotide polymorphism.The authors declare that they have no competing interest.BSD participated in design, selection of included articles, data collection, statistical analysis and wrote the manuscript; LCFP participated in data collection; CBL participated in selection of included articles and wrote the manuscript; KGS data collection and wrote the manuscript, LHSC wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/15/9/prepub


Asthmatics are known to have esophageal hypomotility. Vagal hypofunction and prolonged intra-esophageal acidification cause esophageal hypomotility. The contribution of gastroesophageal reflux (GER) and vagal function to esophageal motility in asthmatics is unclear. We studied the relationship between esophageal motility, GER and vagal function in a cohort of adult asthmatics.Thirty mild, stable asthmatics (ATS criteria) and 30 healthy volunteers underwent 24-hour ambulatory esophageal monitoring, manometry, autonomic function testing and GER symptom assessment. 27 asthmatics underwent gastroscopy. A vagal function score calculated from 3 tests  was correlated with esophageal function parameters.Asthmatics (mean age 34.8 (SD 8.4), 60% female) had more frequent GERD symptoms than controls (mean age 30.9 (SD 7.7), 50% female). 10/27 asthmatics had esophageal mucosal damage, 22 showed hypervagal response, none had a hyperadrenergic response. 14 asthmatics had ineffective esophageal motility. Higher GERD-score asthmatics had significantly fewer peristaltic and more simultaneous contractions than controls, and higher esophageal acid contact times than those with lower scores. All reflux parameters were significantly higher and acid clearance time prolonged in asthmatics than controls . There was no correlation between vagal function score and esophageal function parameters.A cohort of adult asthmatics was found to have peristaltic dysfunction and pathological GER, but otherwise normal esophageal motility. The peristaltic dysfunction seems to be associated with vagal hyperreactivity rather than vagal hypofunction. Asthmatics are known to have an increased prevalence of gastroesophageal reflux disease (GERD) symptoms, esophageal hypomotility and abnormal acid exposure .A study revealed that asthmatics had significantly lower LES pressures . In 34 nReduced motility of the esophagus may be due to vagal hypofunction . Howeveret al. reported that 82% of 104 adult asthmatics had abnormal amounts of acid reflux, more frequent reflux episodes, higher esophageal contact times and longer esophageal acid clearance times  irrespective of presence of symptoms [Esophageal hypomotility may also be secondary to damage from prolonged intra-esophageal acidification. Abnormal reflux is prominent in asthmatics. An endoscopic study of 156 adult asthmatics showed that 39% had evidence of esophagitis or Barrett esophagus . A retroHence, whether esophageal hypomotility in asthmatics is due to vagal hypofunction or secondary to damage from gastro-esophageal reflux (GER) is unclear. The aim of this study was to study the relationship between esophageal motility, GER and vagal function in adult asthmatics.1 following bronchodilator administration) [Thirty consecutive asthmatics who met American Thoracic Society (ATS) guidelines for the definition of asthma (12% improvement and 200 mL increase in FEVtration)  were recThis study was approved by the Ethics and Scientific Review Committee of the Faculty of Medicine of the University of Kelaniya, Ragama, Sri Lanka. All procedures were conducted following written informed consent and conform to the Declaration of Helsinki.All subjects underwent autonomic and esophageal function tests, a GERD symptom assessment and the asthmatics underwent upper gastro-intestinal endoscopy.a) valsalva maneuver produced by sustaining a forced expiration through a mouthpiece connected to a manometer (40 mmHg) lasting 15 s, following a deep inspirationb) heart rate variation with quiet and deep breathing (six breaths per minute)c) heart rate response to standing from supine position.Sympathetic function was assessed by:d) blood pressure response to standing from supine position.For correlation with gastro-intestinal function parameters, a vagal function score was calculated from the results of the three tests. For each test, the result of each individual was ranked on a scale from 1 to 100. The three values were added and averaged for an individual.Both asthmatics and controls were screened by a previously validated GERD screening score assessing frequency and severity of seven common upper gastrointestinal symptoms on a 5-point Likert scale. Symptoms included were 1) heartburn 2) regurgitation 3) upper abdominal or chest pain 4) abdominal distension 5) dysphagia 6) cough and 7) belching. A GERD score was calculated as the sum of the products of frequency and severity of each symptom. A cut of score of ≥ 12.5 was considered as a positive GERD score [UGI endoscopy was performed in 27 asthmatics that consented. The procedure was performed by 2 trained physicians using a fibreoptic endoscope  and findings graded according to modified Savary-Miller criteria . EndoscoEsophageal motility was assessed by the stationary pull-through method, using a water perfused system  according to standard methodology. We had no provision to measure LES pressure using a Sleeve mechanism.Antacids were discontinued 24 hours before, drugs that could affect gastrointestinal motility at least 48 hours before and any anti-secretory drugs one week prior to the study. All asthma medication was withdrawn for 48 hours, allowing inhaled beta-2 agonists on an as needed basis. No food or drink was allowed for at least 6 hours prior to intubation to reduce risk of aspiration during intubation .Nasal intubation was done with application of local anesthetic gel (lignocaine) and a lubricant gel. LES, esophageal body and UES pressures were studied according to standard pull-through method in response to 5 mL water boluses. Overall parameters were summated for each subject to classify them manometrically .24-hour ambulatory esophageal pH monitoring was performed on all subjects using a dual sensor monocrystalline antimony catheter , according to standard methodology . The disth and 95th centile values unless specified otherwise. The upper 95th centile of normal values was used to classify abnormal pH exposure. Categorical variables were compared using the χ2 and Fisher Exact tests. Spearman rank correlation was used to study the association between vagal function and esophageal motility and 24 hour ambulatory pH monitoring variables. A P value of ≤ 0.05 was considered significant. All statistical analysis was performed using SPSS version 10.0 for Windows software .Non parametric tests (Wilcoxon Rank Sum test for paired data and Mann Whitney U test for unpaired data) were used to compare the data between the asthmatics and controls. Data was expressed as median (range) using 5The 30 asthmatics and 30 controls that completed the study were found to be comparable in terms of age, gender and body mass index. The asthmatics had a higher frequency and severity of GERD symptoms Table .All asthmatics had only occasional asthma symptoms over the preceding two weeks before the study. They continued their current medication Table . On GERDTwenty seven asthmatics consented to UGI endoscopy. Of 20 asthmatics with positive GERD symptom scores, 12 had no evidence of mucosal damage, 7 had grade I esophagitis and one subject had grade III esophagitis. Of 7 asthmatics with negative GERD symptom scores, 5 had no evidence of mucosal damage and 2 subjects had grade I esophagitis.The test values of 29 of the controls were within the normal ranges defined for tests of cardiovascular autonomic function . One demDuring the stationary esophageal manometry study, the results of 1 asthmatic and 10 control subjects was excluded from analysis, as equipment failure prevented the manometry study from being performed in these subjects. In those subjects, the position of the lower esophageal sphincter was located by the pH step-up method.According to the current classification of esophageal motility patterns , 16 contP = 0.044, Mann Whitney U test). When the data of the 4 controls with IEM was considered, the difference remained, though not significant. Asthmatics with positive GERD symptom scores had a significantly lower percentage of peristaltic contractions and a significantly higher percentage of simultaneous contractions than the controls .There was no difference of LES mean end-expiratory values and LES relaxation between asthmatics and controls. There was no difference in the LES length or in UES parameters between the two groups. There was no significant difference in the mean peristaltic amplitude and duration for each group at each position  in the esophagus and the mean peristaltic wave velocity between the two groups Table . When thls Table . There wAll reflux parameters were found to be significantly higher in asthmatics compared to controls Table . AsthmatAsthmatics had significantly prolonged proximal and distal acid clearance time  compared to controls . The median acid clearance time values in asthmatics with positive GERD symptom scores was higher than those who had negative GERD symptom scores, but the difference was not significant. The GERD score showed significant negative correlation with LES length and significant positive correlation with the percentage time pH < 4 in the upright position in both proximal and distal positions and the duration of the longest reflux at the proximal sensor. The score also showed an expected negative correlation with amplitude of contractions and UES pressure and expected positive correlations with all the other proximal and distal reflux parameters, though not significant.Asthmatics are known to have a high prevalence of GER and GER is known to trigger asthma . InitialFactors that may promote GER in asthma include, physical characteristics of the individuals , disruption of LES mechanism , reduced LES pressure and increased esophageal acid contact times (effect of asthma medication)  and autoIn this study, the asthmatics demonstrated a high use of oral medication Table . RegressThe asthmatics were recruited from medical clinics. In their history on recruitment it was clarified that these were patients who had been having asthma for a considerable period of time and even childhood asthma. They did not have long term symptoms of gastro-oesophageal reflux disease. The classification regarding presence or absence of GERD symptoms was based on a questionnaire with a recall period of 4 weeks. However, there is no possibility of excluding ‘silent gastro-oesphageal reflux’. So there is a possibility that some may have had GERD as their primary pathology.The present study revealed that 10 of 27 asthmatics (37%) had evidence of esophagitis. The average prevalence of erosive esophagitis in asthmatics reported in a recent systematic review was also 37% . Most stet al. reported pathological reflux in 50% of asthmatic children in the proximal esophagus and in 16% in the proximal esophagus [Twenty two (73.3%) asthmatics had abnormally high acid exposure of the distal esophagus and 20 (66.7%) of the proximal esophagus. Individual pH monitoring parameters were significantly higher  in asthmatics compared to controls. The acid exposure values were higher in asthmatics that scored positive for GERD symptoms compared to those with lower values. Other studies in similar settings to ours  have reported high acid exposure values ranging from 14.8-81.8%. A systematic review of these studies revealed a pooled sample-size weighted average prevalence of 50.9% . Of the sophagus . DeMeestsophagus .The anti-reflux barriers that protect against reflux are the gastresophageal junction (LES function), esophageal body motor function and acid clearance and the UES .Asthmatics have been shown to have lower LES pressures ,32. The Another disadvantage in our methodology and hence a major limitation in our study is that stationary pull-through manometry does not record LES pressure continuously, hence fails to capture sphincter relaxations and intermittent reflux episodes.The UES parameters in our study were similar in asthmatics and controls. Ours was not an ideal setup to measure UES pressure. This is a major limitation in our study, hence data on UES measurement is not discussed here.Esophageal peristalsis  and amplIn the present study the asthmatics demonstrated low peristaltic activity but otherwise normal esophageal manometry. Hence, it is likely that these asthmatics do not have an intrinsic esophageal motility abnormality leading to GER. Therefore, rather than vagal hypofunction contributing to the esophageal dysmotility, as is currently accepted, it may be vagal-hyperreactivity that leads to esophageal dysmotility. Our results lead us to speculate whether this is due to vagal hyperreactivity-induced increased secretion. Whether the reduced esophageal motility is secondary to increased GER due to vagal hyperreactivity and resulting reduced acid clearance warrants further study.When esophageal parameters were grouped according to motility patterns, asthmatics were found to have a higher frequency of abnormal motility patterns. The most common motility pattern among asthmatics was ineffective esophageal motility (IEM) 44.4% of 29 asthmatics). IEM has previously been described as the commonest motility pattern in subjects with reflux-associated respiratory symptoms . Campo eIEM is associated with increased acid clearance times in the distal esophagus . EsophagVagal hypofunction is known to contribute to esophageal hypomotility . StudiesThe current Montreal consensus on GERD states that though frequency and severity of symptoms have been shown to have a moderate correlation with severity of endoscopic findings in several studies, symptoms cannot accurately predict endoscopic findings of an individual patient . In thisFrom the present study findings it seems that hyper-vagal response and prolonged esophageal acid exposure are the more likely reasons for the esophageal peristaltic dysfunction. Increased vagal stimulation increases rate and amount of acid secretion and could therefore augment the damaging effects of GER in these subjects . Acid alIn conclusion, the present study showed that asthmatics with mild, clinically stable asthma have peristaltic dysfunction and increased GER, but otherwise normal esophageal motility parameters compared to healthy controls. Those with more severe GERD symptoms had more peristaltic dysfunction. The asthmatics also showed a vagal hyper-reactivity rather than a vagal hypofunction. Whether the peristaltic dysfunction is secondary to increased GER resulting from the vagal-hyperreactivity warrants further study.1: Forced expiratory volume in first second; GER: Gastro-esophageal reflux; GERD: Gastro-esophageal reflux disease; IEM: Ineffective esophageal motility; LES: Lower esophageal sphincter; TLESR: Transient lower esophageal sphincter relaxation; UGI: Upper gastro-intestinal.ATS: American Thoracic Society; FEVThe authors have no competing interests.All authors were involved in study conception, design, manuscript drafting and revision. AP DS and ASD conducted upper gastro-intestinal endoscopies in asthmatics. DLA and AP were involved in statistical analysis. DLA carried out data acquisition. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/12/140/prepub