Men who have sex with men (MSM) are a high risk population for human immunodeficiency virus (HIV) infection. Our study aims to find whether MSM who were recruited online had a higher prevalence of self-reported unprotected anal intercourse (UAI) than those who were recruited offline.A meta-analysis was conducted from the results of published studies. The analysis was stratified by the participants’ geographic location, the sample size and the date of the last reported UAI.OR) = 1.35, 95% CI = 1.13-1.62, P < 0.01]. The subgroup analysis results also showed that the prevalence of UAI was higher in the European subsample  and in sample sizes of more than 500 individuals  in the online group compared to the offline group. The prevalence of UAI was also significantly higher when the time of the last UAI was during the last 3 or more months  in the online group compared to the offline group. A sensitivity analysis was used to test the reliability of the results, and it reported that the results remained unchanged and had the same estimates after deleting any one of the included studies.Based on fourteen studies, MSM who were recruited online (online-based group) reported that 33.9%  of them had UAI versus 24.9%  of MSM who were recruited offline (offline-based group). The results showed that it is more likely for an online-based MSM group to have UAI with male partners than an offline-based MSM group [odds ratio (A substantial percentage of MSM were recruited online, and they were more inclined to engage in UAI than MSM who were recruited offline. Targeted interventions of HIV prevention programs or services are recommended when designing preventive interventions to be delivered via the Internet. Currently, the prevalence of human immunodeficiency virus (HIV) among men who have sex with men (MSM) is rapidly increasing worldwide-4. MSM ahttp://www.gaydar.net/); they also perceive that this method is convenient and cost-effective because it is private, anonymous, safe and convenient in the process of communication. Although many studies have applied offline-based sampling to MSM[The recruitment of MSM for studies is a challenge for researchers because no sampling frame exists for MSM and public acknowledgement of membership may also be stigmatized in some cases. The Intg to MSM-19, seveg to MSM-22 samplSome studies have shown that online-based MSM were more likely to report different socio-demographic profiles and riskIt is important to examine the validity of sampling online compared to more established venue-based methods because the individuals recruited by sampling online may be fundamentally different from those recruited offline, with respect to their sexual risks. In the past few decades, several studies,28-31 haStudies published before December 2013 that examined the prevalence of UAI among MSM were carefully selected from the following databases: PubMed (1966 to 2013), Springer (1996 to 2013), Cochrane Library (1993 to 2013), Google Scholar (1987 to 2013), China National Knowledge Infrastructure  and the Wanfang database . The date of the last search was February 8, 2014. The databases were searched using the following key words: “men who have sex with men”, “Internet”, “web”, “online”, “offline”, “venue”, “MSM”, “gay”, “homosexuality”, “risky behavior”, “sexual behavior” or “anal intercourse”. No language restrictions were carried out for this study. All of the studies that investigated the prevalence of UAI among online-based MSM or offline-based MSM were evaluated carefully. The selection criteria are listed as follows: (1) the reports were full-length, published papers; (2) the studies reported data for UAI among MSM, and the duration of UAI was not limited; and (3) all of the studies recruited MSM both online and offline. We excluded the studies in which the reported UAI data were from MSM recruited either only online or offline and any meeting or conference abstract data.Two investigators (Yang ZR and Zhang SC) reviewed the abstracts independently to determine whether the studies conformed to the eligibility criteria for this study. Two other investigators (Jin MH and Dong ZQ) reviewed the references in the papers to identify any additional studies. A third investigator (Han JK) carried out additional assessments if discrepancies were generated. In our study, there were no discrepancies.The data items included study details , characteristics of participants , and different risky sexual behaviors . Two investigators (Yang ZR and Dong ZQ) extracted the data independently using the standardized protocol by coding forms, and a third investigator (Jin MH) reviewed the results.For each study, we recorded the first author’s name, the publication year, the country and geographic location, the definition of UAI , the sample size and the prevalence of UAI. UAI was defined as having never used a condom during anal sex in the last year, using condoms inconsistently in the process of anal intercourse in the last year, UAI with a serodiscordant or HIV-unknown male partner, UAI with a male partner in the last three months, UAI in the last two months, UAI with any male partner in the last three months, UAI with a casual partner in the last six months, UAI in the last three months, UAI with a male partner in the last incident of anal sex, any UAI with any partner, or being engaged in any MSM UAI in the last year.This study assessed the comparison of the UAI prevalence between MSM who were recruited online (online-based group) vs. those who were recruited offline (offline-based group). The online-based group’s participants were investigated through the Internet (online), and the respondents of the offline-based group were surveyed in bathhouses, bars, clubs, etc. We examined the association with the prevalence of UAI among MSM between the online-based group and the offline-based group. Then, we merged data from the same geographic location, sample size and the last incidence of UAI by means of subgroup analysis.ORs) and 95% confidence intervals (95% CI) were calculated with respect to each study. We quantified the effect of heterogeneity by means of the formula I2 = 100% × (Q − df)/Q[Q-statistic[Q-statistic (P < 0.10) existed; otherwise, the fixed effect model was used[The odds ratios (Q − df)/Q. We alsotatistic. The ranwas used.OR, or the pooled estimate of risk, was obtained using the Mantel-Haenszel method in the fixed effect model[OR in the meta-analysis was calculated by weighting the individual ORs using the inverse of their variance[OR was determined using a Z-test[The overall ct model and usinct model. The poovariance. The siga Z-test. To testOR[We measured the asymmetry of the funnel plot by using Egger’s linear regression, which aOR. The intOR.http://tech.cochrane.org/revman) and the STATA software package v.11.0 . All the P values were two-sided. Differences were considered statistically significant if the P value was less than 0.05.The meta-analysis was conducted with Review Manager 5.1 software . The flow chart of study enrollment for this meta-analysis is shown in Figure A total of 14 studies published between 2000 and 2012 were included in this meta-analysis. The characteristics of the selected studies are listed in Table OR was 1.35  for UAI between the online-based group and the offline-based group using the random effect model because between-study heterogeneity was significant .The summary of the prevalence of UAI among MSM is shown in Table OR = 1.38, 95% CI = 1.17-1.63, P < 0.01) and in sample sizes of more than 500 individuals  in the online group compared to the offline group. The prevalence of UAI was also significantly higher when the time of the last UAI was during the last 3 or more months  in the online group compared to the offline group.We performed subgroup analyses stratified by the participants’ geographic location, the sample size and the time of last UAI in this study Table . The subThe effect of any single research study on the overall meta-analysis was carried out by deleting one study at a time. The exclusion of any individual study did not make a significant difference to this meta-analysis, suggesting that the results of our study are statistically reliable.The Egger’s linear regression test was evaluated for funnel plot asymmetry Table . The resWe extracted fourteen studies that included 17,580 MSM who were recruited online (online group) and 10,853 MSM who were recruited offline (offline group) to evaluate the prevalence of UAI among MSM in this study. Our meta-analysis suggested that there was a higher prevalence of UAI among MSM who were recruited online than those recruited offline. The prevalence of UAI was defined differently in the fourteen studies of this meta-analysis, with the prevalence varying between 9.8% and 59.9% among MSM who were recruited online and between 7.5% and 64.9% among those who were recruited offline. These results suggest that the populations under study and the evaluation of risk groups vary in different, we should lead to caution when interpretating differences based on the mode of recruitment.We performed subgroup analyses stratified by the participants’ geographic location , sample size  and the time of last UAI (UAI in the last six or more months and UAI in the last three or less months) to decrease the differences based on the mode of recruitment. The subgroup analyses showed that MSM who were recruited online were associated with increased UAI in studies with a sample size of more than 500 individuals but not in studies with a cohort of less than or equal to 500; this may have been due to reduced sampling bias in studies with larger samples. In addition, a high rate of UAI is primarily due to the definition or scope of UAI; therefore, if the definition or scope of UAI is broader (a broader definition of UAI means that the scope of UAI is unlimited or less limited), the UAI rate would be accordingly higher. Recently, a meta-analysis demonstrSome limitations of this study should be discussed as well. First, only published studies were included in the present meta-analysis. Thus, pOur study found that MSM who were recruited online are more likely to engage in UAI with male partners than offline-based MSM, so data obtained from MSM using convenient Internet networks can provide potentially powerful tools for informing public health interventions. Most of the previous studies were conducted using offline-recruited (venue-based) sampling methods among MSM. Researchers used the results of those studies to design intervention measures targeting ordinary MSM, and these measures may not be suitable for online-based MSM who seek sexual partners. The IntSpecific Internet related interventions may be helpful for online-based MSM, and it is crucial for HIV/STD control and prevention staff to pay more attention to this population to increase the awareness of self-protection and decrease the risk of HIV/STDs among online-based MSM.Increased sexual risk behavior has been linked to MSM who seek their partners online. This new situation will have extremely important implications for worldwide HIV/AIDS prevention, and increased attention should be paid to MSM because of this association. This study supports the results that a substantial percentage of MSM who were recruited online are more likely to engage in UAI than MSM who were recruited offline. Because we only used published papers in this study, we also need to pay close attention to the influences of unpublished studies, such as dissertations and papers presented at various conferences, which may help to confirm the results of this study. Targeted interventions of HIV prevention programs or services are recommended when designing preventive interventions to be delivered through the Internet.HIV: Human immunodeficiency virus; MSM: Men who have sex with men; UAI: Unprotected anal intercourse; STD: Sexually transmitted diseases; CNKI: China National Knowledge Infrastructure.The authors declare that they have no competing interests.JM, HJ and DZ participated in the design of the study and data collection. YZ performed the statistical analysis. YZ and ZS conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/508/prepub


TNF) –308G>A and nitric oxide synthase 3 (NOS3) +894G>T polymorphisms and migraine. We performed a meta-analysis of case-control studies to evaluate whether the TNF –308G>A and NOS3 +894G>T polymorphisms confer genetic susceptibility to migraine.Conflicting data have been reported on the association between tumor necrosis factor  and 95% confidence intervals (95% CI) assuming allele contrast, dominant model, recessive model, and co-dominant model as pooled effect estimates.We performed an updated meta-analysis for TNF –308G>A and six studies in 1055 migraineurs and 877 controls for NOS3 +894G>T were included in the analysis. Neither indicated overall associations between gene polymorphisms and migraine risk. Subgroup analyses suggested that the “A” allele of the TNF –308G>A variant increases the risk of migraine among non-Caucasians . The risk of migraine with aura (MA) was increased among both Caucasians and non-Caucasians. Subgroup analyses suggested that the “T” allele of the NOS3 +894G>T variant increases the risk of migraine among non-Caucasians .Eleven studies in 6682 migraineurs and 22591 controls for TNF –308G>A polymorphism may act as a genetic susceptibility factor for migraine among non-Caucasians and that the NOS3 +894G>T polymorphism may modulate the risk of migraine among non-Caucasians.Our findings appear to support the hypothesis that the  Migraine is characterized by recurrent, moderate to severe, throbbing headache attacks, associated with photophobia, phonophobia, nausea, and vomiting. Migraine is remarkably common, affecting approximately 11% of the adult population around the world . This coThe etiology of migraine is complex, involving both multiple genetic and environmental factors . It is nSterile meningeal inflammation is a key mechanism that may underlie the sustained activation and sensitization of perivascular meningeal nociceptors. Cytokines and nitric oxide (NO) have been implicated in the pathogenesis of migraine in both animal and human studies .Tumor necrosis factor-alpha (TNF-α) is an important cytokine that can promote powerful hyperalgesia by causing prostanoid release, increasing nerve growth factor (NGF) and bradykinin receptor expression, or by modulation of activity within sympathetic fibers. Several studies have indicated changes in serum , plasma NO is a potent endogenous vasodilator, which is a key molecule affecting the pain associated with migraine , as it wl-arginine and molecular oxygen by the NO synthase (NOS) family. In endothelial cells, NO is synthesized by the endothelial NOS (eNOS). Deregulation of eNOS influences the plasma level of NO, which has an anti-inflammatory effect. The haplotype of the NOS3 gene is associated with variability in endogenous NO formation  OR “migraine disorders” [All Fields] OR “migraine” [All Fields] OR “migraine disorder” [All Fields] OR “headache disorders” [MeSH Terms] OR “headache disorders” [All Fields] OR “headache” [All Fields] OR “headache disorder” [All Fields])) OR “migraine with aura” [MeSH Terms] OR “migraine with aura” [All Fields] OR “migraine without aura” [MeSH Terms] OR “migraine without aura” [All Fields]) AND  AND  OR “variant” [All Fields] OR “variants” [All Fields] OR “rs1800629” OR “TNF –308G>A”) AND “humans” [MeSH Terms]. In addition, the reference lists of selected papers and potentially relevant studies in the previous meta-analyses were also screened to further identify potentially relevant papers through reference association. The last search was updated in July 2014. The selection process is shown as a flow chart in Two of the authors (MC and WT) searched the PubMed, EMBASE, Science Citation Index (SCI), and Cochrane Library electronic databases for tumor necrosis factor and endothelial nitric oxide synthase  AND “headache” OR “headache disorder” OR “migraine” OR “migraine disorder” OR “migraine with aura” OR “migraine without aura” AND “gene” OR “polymorphism” OR “genetic variation” OR “polymorphisms” OR “rs1800629” OR TNF –308G>A or NOS3 +894G>T polymorphism and migraine risk based on a case-control or cohort design, and (ii) that contained information on the sample sizes, distribution of genotypes, and allele frequencies for the polymorphisms investigated among migraineurs and non-migraineurs allowing for calculation of crude risks for migraine. In publications with overlapping cases and/or controls, the most recent or largest population was chosen.The meta-analysis included only articles (i) that evaluated the association of the In the first step of the analysis, two of the investigators (MC and WT) independently identified all studies not meeting any of the pre-specified criteria by screening the titles and abstracts, and these studies were excluded. In the second step, the same investigators independently evaluated the full-text publications of the remaining studies. Studies were excluded if they did not meet all of the criteria. Any discrepancies were adjudicated by a third reviewer (ZD).Studies were excluded if one of the following existed: (1) studies with insufficient genotyping data of the participants; (2) case-only studies; (3) review articles; (4) animal studies; (5) editorials; (6) abstracts; (7) studies that reported associations between gene polymorphisms and recovery from a migraine attack; (8) studies that reported associations between gene polymorphisms and response to any therapy; (9) studies that deviated from Hardy—Weinberg equilibrium.Two of the investigators (MC and WT) independently reviewed, extracted, and entered data from the studies in a standardized data extraction form. Discrepancies were resolved by consensus. The items recorded were authors’ names, year of publication, ethnicity of subjects, study design, genotyping method, migraine status , diagnostic criterion of cases, gender of individuals in the study population, study size, allele and genotype frequencies, and additional potentially relevant information. We attempted to collect genetic information for all migraineurs and migraineurs with and without aura separately as well as for the whole study population and for females and males separately. If not presented in the article, allele and genotype frequencies were calculated when possible. For studies that did not allow extraction of all relevant information, including genotype and allele frequencies, we e-mailed the corresponding authors to obtain the missing information. Authors not responding within 1 week were sent up to two reminders by e-mail.http://www.ohri.ca/programs/clinical_epidemiology/oxford.asp). The quality of each study was assessed and awarded stars based on indicators of quality, including selection of study population, comparability, and exposure assessment independently. The quality score ranged from 0 (worst) to 10 points (best). Any discrepancies were adjudicated by a third reviewer (LH).The quality of studies was assessed by the same two reviewers (MC and WT) independently using a modified 10-point Newcastle—Ottawa Scale (NOS) , dominant model , recessive model , and allele contrast . Heterogeneity across studies was detected by the χ2 test-based Q-statistic (P < 0.10 was deemed as evidence of heterogeneity) as well as I2 statistic, which takes values between 0% and 100%  [All analyses were performed using STATA version 12.0 . Unless otherwise stated, P < 0.05 was taken to indicate statistical significance. Hardy—Weinberg equilibrium (HWE) for each study was determined using the goodness-of-fit geneity) . When thgeneity)  was applgeneity)  was perfTNF –308G>A [NOS3 +894G>T [TNF –308G>A and NOS3 +894G>T [TNF –308G>A, we excluded one abstract [NOS3 +894G>T, one was excluded because no information about the distribution of genotypes was provided [TNF –308G>A and NOS3 +894G>T [TNF –308G>A was available but not that for NOS3 +894G>T. MC and WT independently read the articles, and 11 studies were finally included for TNF –308G>A and 6 studies were included for NOS3 +894G>T. Kappa statistic for agreement of TNF –308G>A between the two reviewers was 0.9, and that for NOS3 +894G>T was 0.86. Any discrepancies were adjudicated by a third reviewer (ZD). –308G>A , 44–46,  +894G>T , 47–52,  +894G>T . After fabstract . Anotherabstract . Among t +894G>T , informaTNF –308G>A, 7 [NOS3 +894G>T, six reported genotype and allele frequencies for all participants and for overall migraine as well as those stratified by aura [Among the 11 papers focusing on 08G>A, 7 , 37, 44 08G>A, 7 , 45. Aft08G>A, 7 . Among t by aura –51, 53,  by aura , 47. We TNF –308G>A polymorphisms [NOS3 +894G>T polymorphisms [The detailed characteristics of each study included in the meta-analysis are presented in orphisms , 44, 45 orphisms , 55, 56.TNF –308G>A, all studies were of a case-control design [NOS3 +894G>T, all studies were of a case-control design, and all studies were performed in adults. Three studies were performed in Caucasians [For l design , 44–46 el design . One stul design , and thel design , 45, 46.l design , 36, 46 l design , 34, 45.ucasians , 53, 56 ucasians , 51, 55.TNF –308G>A and NOS3 +894G>T among migraineurs and controls are listed in The distributions of genotypes and alleles for TNF –308G>A and NOS3 +894G>T polymorphisms, respectively.Tables TNF –308G>A with 6682 genotyped migraine cases and 22591 controls, no significant association between TNF –308G>A polymorphism and migraine risk was observed under any of the genetic models. Strikingly, the meta-analysis provided an OR of 1.74  under A vs. G, an OR of 1.82  under AA+GA vs. GG, and an OR of 1.78  under GA vs. GG among non-Caucasians, while none of the contrast models showed a significant association in Caucasian populations , as shown in By pooling the six selected studies with 1055 genotyped migraine cases and 877 controls for TNF –308G>A polymorphism and MA . Nevertheless, the reverse effects were observed in studies of the TNF –308G>A polymorphism and migraine without aura (MO). Using Galbraith plots, we identified individual studies as important sources of heterogeneity [There was no significant heterogeneity among studies of ogeneity , 51, as ogeneity , 30, 31.TNF –308G>A, P = 0.707 and 0.508, respectively, for NOS3 +894G>T)  Fig  and 6B.The overall results of this meta-analysis did not indicate associations between any of the polymorphisms investigated and migraine. However, the associations differed by ethnicity and clinical phenotype.TNF –308G>A polymorphism, stratified analysis by ethnicity suggested an increased risk of migraine among non-Caucasians. When stratifying the data by type of migraine, we observed an increased risk of MA among both non-Caucasians and Caucasians, but especially among non-Caucasians.Among studies investigating the NOS3 +894G>T variant increased the risk of migraine among non-Caucasians, which was driven by associations for MA. Assuming an allele T frequency of 10.64% among controls and using the TT vs. GT+GG genetic model, this study had about 84.3% power at a significance level of α = 0.05 to detect an effect size of 1.84 among non-Caucasians [Subgroup analyses suggested that the T allele of ucasians .NOS3 +894G>T genotype GT have an increased risk of allodynia [“Neurogenic inflammation” around the dural trigeminal afferents is known to play an important role in the generation of migraine attacks  and to cllodynia .TNF promoter has been shown to be associated with a high level of TNF-α expression [HLA alleles, which have high linkage disequilibrium with TNF. Individuals carrying the “A” allele as part of the HLA Al, B8, DR3 haplotypes, which are population-specific within the human MHC region [The levels of TNF-α and NO are under genetic control. An “A” at position –308 in the pression , 62. A fC region , may havC region , in whicTNF –308G>A and NOS3 +894G>T polymorphisms with predisposition to migraine have been examined in a number of studies, which reported inconsistent results. Due to the failure of independent association studies in identifying a role for the TNF –308G>A polymorphism in migraine, two meta-analyses were performed to reach definitive and reliable conclusions. Gu et al. [TNF –308G>A polymorphism with migraine based on six studies in Asian populations (985 cases and 958 controls); their results indicated that the TNF –308G>A polymorphism was associated with migraine risk in Asians. Subgroup analysis suggested that there was a statistically significant result for MA but not for MO. Another meta-analysis conducted by Schuerks et al. [TNF –308G>A polymorphism indicated no overall association with migraine. Subgroup analyses suggested that the “A” allele of the TNF –308G>A variant increases the risk of migraine among non-Caucasian populations, which was driven by associations for MO. In our study, subgroup analyses suggested that the risk of migraine with aura (MA) was increased among both Caucasians and non-Caucasians. Performance of a genome-wide association study (GWAS) enables identification of genetic variants in many multi-pathogenic diseases. To date, neither the TNF –308G>A nor the NOS3 +894G>T polymorphism has been included in GWAS.The associations of the u et al.  performes et al.  based onTNF –308G>A or NOS3 +894G>T polymorphism and migraine risk. There are two possible explanations for the observed overall lack of associations. Either, there may be really be no association and the positive results in some studies may have occurred by chance or due to certain study characteristics, or the pattern of association may be more complex and involve additional factors. In this context, considerations such as the heterogeneity among studies, different sources of controls, and variable sample sizes among the studies must be addressed.Despite their plausible associations with migraine, this meta-analysis revealed no general associations between the TNF –308G>A polymorphism with migraine and MO, which may have been due to differences between subgroups. Furthermore, assuming that the frequency of the “A” allele is 2.74% among controls and using the A vs. G genetic model, this study had only about 61.5% power at a significance level of = 0.05 to detect an effect size of 1.74 among non-Caucasians [NOS3 +894G>T, not all information was available, which would have been valuable to draw clear associations with migraine among populations of different ethnicities.Specifically, there were moderate to high degrees of heterogeneity among the studies investigating the association of the ucasians . To achiInherent bias, such as sampling bias, selection bias, publication bias, and within-study bias, cannot be avoided in a meta-analysis of observational studies. The following biases must be taken into consideration when interpreting the results of the present study. First, all of the studies included in the meta-analysis were published in English, so selection bias may have occurred. Second, we did not include fugitive literature, including academic dissertations and conference papers, so there may have been bias in provision of data. Third, there was a lack of unified sources of controls. The controls were selected from three sources: hospital-based, healthy population, and family members of the migraineurs, so selection bias may have occurred especially among the studies with hospital-based controls. Fourth, there was heterogeneity across the studies, which may have masked significance in the present study. Another limitation of our study was that we did not analyze the gene-to-gene or gene-environment interactions that may contribute to modification of migraine risk due to a lack of data among the existing publications eligible for this meta-analysis.TNF –308G>A polymorphism in the TNF region may represent a risk for migraine among non-Caucasians and MA among both Caucasians and non-Caucasians. The NOS3 +894G>T polymorphism of eNOS may represent a risk for migraine among non-Caucasians. However, given the heterogeneity among study designs, the results of this meta-analysis should be interpreted with caution. Further cohort studies based on pedigree are warranted to evaluate population-specific effects, including population stratification.In summary, the general results of our meta-analysis do not support a strong overall effect, but provide statistical evidence that the S1 Checklist(DOCX)Click here for additional data file.S2 Checklist(DOC)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S1 FigTNF –308G>A. B: S1B Fig Galbraith plot for NOS3 +894G>T.A: S1A Fig Galbraith plot for (EPS)Click here for additional data file.S1 TableMA: migraine with aura; MO: migraine without aura(DOCX)Click here for additional data file.S2 TableMA: migraine with aura; MO: migraine without aura(DOCX)Click here for additional data file.


In a systematic review and meta-analysis, Giovanni Musso and colleagues examine the association between non-alcoholic fatty liver disease and chronic kidney disease.Please see later in the article for the Editors' Summary Chronic kidney disease (CKD) is a frequent, under-recognized condition and a risk factor for renal failure and cardiovascular disease. Increasing evidence connects non-alcoholic fatty liver disease (NAFLD) to CKD. We conducted a meta-analysis to determine whether the presence and severity of NAFLD are associated with the presence and severity of CKD.2 or proteinuria were included. Two reviewers extracted studies independently and in duplicate. Individual participant data (IPD) were solicited from all selected studies. Studies providing IPD were combined with studies providing only aggregate data with the two-stage method. Main outcomes were pooled using random-effects models. Sensitivity and subgroup analyses were used to explore sources of heterogeneity and the effect of potential confounders. The influences of age, whole-body/abdominal obesity, homeostasis model of insulin resistance (HOMA-IR), and duration of follow-up on effect estimates were assessed by meta-regression. Thirty-three studies  were included. For 20 studies , we obtained IPD. NAFLD was associated with an increased risk of prevalent  and incident  CKD. Non-alcoholic steatohepatitis (NASH) was associated with a higher prevalence  and incidence  of CKD than simple steatosis. Advanced fibrosis was associated with a higher prevalence  and incidence  of CKD than non-advanced fibrosis. In all analyses, the magnitude and direction of effects remained unaffected by diabetes status, after adjustment for other risk factors, and in other subgroup and meta-regression analyses. In cross-sectional and longitudinal studies, the severity of NAFLD was positively associated with CKD stages. Limitations of analysis are the relatively small size of studies utilizing liver histology and the suboptimal sensitivity of ultrasound and biochemistry for NAFLD detection in population-based studies.English and non-English articles from international online databases from 1980 through January 31, 2014 were searched. Observational studies assessing NAFLD by histology, imaging, or biochemistry and defining CKD as either estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 mThe presence and severity of NAFLD are associated with an increased risk and severity of CKD.Please see later in the article for the Editors' Summary Chronic kidney disease (CKD)—the gradual loss of kidney function—is becoming increasingly common. In the US, for example, more than 10% of the adult population (about 26 million people) and more than 25% of individuals older than 65 years have CKD. Throughout life, the kidneys perform the essential task of filtering waste products  and excess water from the blood to make urine. CKD gradually destroys the kidneys' filtration units, the rate of blood filtration decreases, and dangerous amounts of waste products build up in the blood. Symptoms of CKD, which rarely occur until the disease is very advanced, include tiredness, swollen feet, and frequent urination, particularly at night. There is no cure for CKD, but progression of the disease can be slowed by controlling high blood pressure and diabetes (two risk factors for CKD), and by adopting a healthy lifestyle. The same interventions also reduce the chances of CKD developing in the first place.CKD is associated with an increased risk of end-stage renal (kidney) disease and of cardiovascular disease. These life-threatening complications are potentially preventable through early identification and treatment of CKD. Because early recognition of CKD has the potential to reduce its health-related burden, the search is on for new modifiable risk factors for CKD. One possible new risk factor is non-alcoholic fatty liver disease (NAFLD), which, like CKD is becoming increasingly common. Healthy livers contain little or no fat but, in the US, 30% of the general adult population and up to 70% of patients who are obese or have diabetes have some degree of NAFLD, which ranges in severity from simple fatty liver (steatosis), through non-alcoholic steatohepatitis (NASH), to NASH with fibrosis (scarring of the liver) and finally cirrhosis (extensive scarring). In this systematic review and meta-analysis, the researchers investigate whether NAFLD is a risk factor for CKD by looking for an association between the two conditions. A systematic review identifies all the research on a given topic using predefined criteria, meta-analysis uses statistical methods to combine the results of several studies.The researchers identified 33 studies that assessed NAFLD and CKD in nearly 64,000 participants, including 20 cross-sectional studies in which participants were assessed for NAFLD and CKD at a single time point and 13 longitudinal studies in which participants were assessed for NAFLD and then followed up to see whether they subsequently developed CKD. Meta-analysis of the data from the cross-sectional studies indicated that NAFLD was associated with a 2-fold increased risk of prevalent (pre-existing) CKD . Meta-analysis of data from the longitudinal studies indicated that NAFLD was associated with a nearly 2-fold increased risk of incident (new) CKD . NASH was associated with a higher prevalence and incidence of CKD than simple steatosis. Similarly, advanced fibrosis was associated with a higher prevalence and incidence of CKD than non-advanced fibrosis.These findings suggest that NAFLD is associated with an increased prevalence and incidence of CKD and that increased severity of liver disease is associated with an increased risk and severity of CKD. Because these associations persist after allowing for established risk factors for CKD, these findings identify NAFLD as an independent CKD risk factor. Certain aspects of the studies included in this meta-analysis  and the methods used in the meta-analysis may limit the accuracy of these findings. Nevertheless, these findings suggest that individuals with NAFLD should be screened for CKD even in the absence of other risk factors for the disease, and that better treatment of NAFLD may help to prevent CKD.http://dx.doi.org/10.1371/journal.pmed.1001680.Please access these websites via the online version of this summary at US National Kidney and Urologic Diseases Information Clearinghouse provides information about all aspects of kidney disease; the US National Digestive Diseases Information Clearinghouse provides information about non-alcoholic liver diseaseThe US National Kidney Disease Education Program provides resources to help improve the understanding, detection, and management of kidney disease (in English and Spanish)The chronic kidney disease, including some personal stories, and information on non-alcoholic fatty liver diseaseThe UK National Health Service Choices website provides information for patients on chronic kidney disease (in English and Spanish)The US National Kidney Foundation, a not-for-profit organization, provides information about UK National Kidney Federation provides support and information for patients with kidney disease and for their carersThe not-for-profit non-alcoholic fatty liver disease, including a patient storyThe British Liver Trust, a not-for-profit organization, provides information about  Chronic kidney disease (CKD) affects 4%–13% of the Western adult population and over 25% of individuals older than 65 years Early recognition and treatment of CKD aimed at reducing renal disease progression and CVD complications may limit its health-related burden The high morbidity, mortality, and health care costs associated with CKD have led investigators to seek novel modifiable risk factors. Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, affects 30% of the general adult population and up to 60%–70% of diabetic and obese patients We searched English and non-English language publications on MEDLINE, Ovid MEDLINE In-Process, EMBASE, ISI Web of Science, and Cochrane Library, and abstracts from the annual American Association for the Study of Liver Disease (AASLD), the American Gastroenterological Association (AGA), the European Association for the Study of the Liver (EASL), the Digestive Disease Week (DDW), and the American Society of Nephrology (ASN) Kidney Week meetings from 1980 through January 31, 2014. Search terms were: chronic kidney disease OR CKD OR kidney function OR kidney failure OR renal disease OR renal insufficiency OR renal failure OR glomerular filtration rate (GFR) OR estimated glomerular filtration rate (eGFR) OR creatinine OR albuminuria OR microalbuminuria OR macroalbuminuria OR proteinuria OR kidney injury AND NASH OR NAFLD OR non-alcoholic steatohepatitis OR non-alcoholic fatty liver disease OR fatty liver OR liver fat OR steatosis OR liver enzymes OR transaminase OR ALT OR AST OR GGT OR severity of liver disease OR fibrosis. A full list of the search strategies in different databases is reported in 2, as estimated using the creatinine-based Modified Diet in Renal Disease (MDRD) or Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations 2 using 24-hour urinary studies, (3) persistent (>3 months) kidney damage (regardless of GFR), as defined by proteinuria , (4) other abnormalities due to tubular disorders or structural abnormalities detected by electrolyte or urinary sediment alterations, histology, imaging, or (5) history of kidney transplantation Criteria were observational studies including adult (age ≥18 y) populations of any sex or ethnicity, with a diagnosis of NAFLD and CKD. NAFLD had to be diagnosed by (1) liver histology, (2) imaging , or (3) biochemistry . Competing causes of steatosis, including alcohol consumption and viral hepatitis infection had to be excluded according to standard guidelines per se on GFR).Excluded from the meta-analysis were nnon-human studies, letters/case reports, studies including fewer than ten individuals, articles not reporting outcomes of interest or primary data , or using inadequate case definitions. In particular, studies were excluded that did not adequately consider competing causes of hepatic steatosis including alcohol, or viral hepatitis, or that enrolled a mixed population of cirrhotic and non-cirrhotic individuals , and CKD stage 5 (eGFR<15 ml/min/1.73 m2) The severity of CKD was the secondary outcome measure. We estimated the effect of the severity of liver disease in NAFLD, as defined by NASH or advanced fibrosis, on the stage of CKD. CKD stage was categorized by GFR according to recent guidelines into CKD stage 3b , using a predefined protocol  was solicited from principal investigators (PIs). PIs were asked to provide the most complete and updated data, even if the follow-up was longer than that used for their respective publications. The quality of the submitted IPD was assessed using pre-specified methods  into a pooled effect measure using the two-stage method In reducing IPD to AD, for dichotomous outcomes we used multivariate logistic regression in cross-sectional studies to obtain log odds ratio (OR) with its standard error (SE), and Cox proportional hazard model in longitudinal studies  to obtain log hazard ratio (HR) and its SE separately for each study. We then combined individual ORs  and HRs  and their 95% CIs from all included studies. Associations with continuous outcome variables were expressed as weighted mean differences (WMD) with 95% CI. Only the most adjusted risk estimates that were reported in the studies were included in the analysis. All measures of dispersion were converted to standard deviations (SDs).p = 0.05.The study-specific risk estimates were pooled using random-effects model, because this approach provides a more conservative assessment of the average effect size than fixed-effects model. Significance was set at 2 statistic and its 95% CI 2 values ≥50%, we explored individual study characteristics and those of subgroups of the main body of evidence.The IWe separately analyzed cross-sectional and longitudinal studies. Furthermore, for each outcome, the results of studies defining NAFLD by histology, imaging, or liver enzyme elevation are presented separately.a priori. These subgroup analyses included repeated analysis after excluding studies not fulfilling each STROBE item, and separate analyses for the following items: diabetes: we examined the effect of NAFLD on CKD in non-diabetic versus diabetic individuals, to assess if the presence of diabetes affects the association of NAFLD with CKD; studies simultaneously adjusting versus studies not adjusting for all the following risk factors for CKD: age, body mass index (BMI), metabolic syndrome , hypertension, smoking status; study design: population-based versus hospital-based; ethnicity (Asian versus non-Asian population), as defined by the investigators. As highlighted by the recent report of the Third Asian Forum of Chronic Kidney Disease Initiatives, there are striking differences in risk factors for CKD between Asian ancestry and the remaining ethnicities: as an example, chronic glomerulonephritis due to IgA nephropathy is among the three leading causes of CKD in Asians, while it is far less common in the rest of the world. Different susceptibility loci involved in innate and adaptive immunity have been identified in recent genome-wide association studies (GWAS), but also environmental factors like infections and regular consumption of herbal remedies, may underlie these epidemiological differences Sensitivity analysis was performed by repeating the meta-analysis after one study at a time was removed to assess whether any one study significantly affected pooled estimates. Additionally, a number of subgroup analyses were planned We therefore explored whether differences in epidemiology of NAFLD and CKD between Asian and non-Asian populations affect the association of NAFLD with CKD studies including exclusively non-cirrhotic patients versus studies including exclusively cirrhotic patients; methods used to estimate GFR; outcomes related to CKD: studies assessing both eGFR and proteinuria versus studies assessing solely eGFR or proteinuria; study data availability: studies providing IPD versus studies providing exclusively AD.When eight or more comparisons were available, the effect of continuous variables including age, whole-body and abdominal obesity  Small study bias was examined by constructing funnel plots and by performing the Egger's test and the trim-and-fill analysis Additionally, for the primary end-point we separately performed a one-stage meta-analysis of studies providing IPD, to examine how the association of NAFLD with CKD was altered when individual patient level covariates were accounted for We used RevMan 5.2 (Nordic Cochrane Center) and SAS 9.2 (SAS Institute) for additional analyses that could not be done with RevMan. The trim-and-fill analysis was performed with Comprehensive Meta-analysis 2.0 (Biostat).The mean (standard deviation [SD]) agreement between the two reviewers for study selection and for quality assessment were 0.89 (0.02) and 0.91 (0.04), respectively. The flow of study selection is reported in Thirty-three studies  were included  and 2 ,) Twelve studies enrolled exclusively non-diabetic individuals Twenty-eight studies  adjusted for potential confounders, including all of the following: age, BMI, metabolic syndrome , hypertension, and smoking , and 3 , N-comparisons = 17, p<0.00001)   . The mag0.00001) . Heterog0.00001) .2 = 0% [95% CI 0%–18%], n comparisons = 13, p<0.00001)  . There wIn both cross-sectional and longitudinal studies, the difference between NAFLD and non-NAFLD patients remained statistically significant even when considering eGFR as a continuous variable or when considering only proteinuria as outcome , BMI , waist circumference , HOMA-IR index , and duration of follow-up .In both cross-sectional and longitudinal studies, meta-regression analysis found no association between CKD and age    . Pooled 0.00001) . There wNASH and advanced fibrosis were also associated with higher ORs for proteinuria and with a lower eGFR than steatosis and non-advanced fibrosis, respectively , BMI , waist circumference , and HOMA-IR index .Meta-regression analysis found no association between CKD and age    . Pooled 0.00001) . There wNASH and advanced fibrosis were also associated with a higher OR for incident proteinuria and with more severe eGFR reduction than steatosis and non-advanced fibrosis, respectively , BMI , waist circumference , HOMA-IR index  and duration of follow-up .Meta-regression analysis found no association between CKD and age    or renal failure (CKD stage 5), was an exclusion criterion in cross-sectional studies, which focused on the association of NAFLD with clinically unrecognized (stage 1–3) CKD.2 = 0% [95% CI 0%–21%], n-comparisons = 7, p = 0.01); OR for CKD stage 4: 3.45 ; OR for CKD stage 5: 3.87   was significantly higher in NASH versus steatosis: OR for CKD stage 3b: 2.49   and 7.2 = 23% [95% CI 0%–35%], n-comparisons = 7, p<0.0001); OR for CKD stage 4: 7.66 ; OR for CKD stage 5: 12.67   was significantly higher in advanced versus non-advanced fibrosis: OR for CKD stage 3b: 7.48   and 8.There was no heterogeneity in the meta-analysis of overall events, suggesting a consistent disease effect.The magnitude and direction of the associations were unaltered across studies fulfilling different STROBE score items in non-diabetic individuals  were included in this analysis. We first analyzed the influence of each single pre-specified individual patient level covariate on the association of NAFLD with CKD with NAFLD and covariate as fixed-effect and the study as random-effects. In a second step, we did a complete case multivariable analysis with respect to NAFLD and all pre-specified covariates. The covariates entered in the models were age, BMI, metabolic syndrome (presence versus absence), diabetes (presence versus absence), hypertension (presence versus absence), smoking status (current smokers versus non-smokers), ethnicity (Asian versus non-Asian population), cirrhosis (presence versus absence), waist circumference, HOMA-index, duration of follow-up .The magnitude of the effect of NAFLD on CKD remained largely unaffected after adjusting for the covariates separately and in the fully adjusted models .The main results of our analysis are the following: (1) NAFLD was associated with an increased prevalence and incidence of CKD; (2) liver disease severity in NAFLD was associated with an increased risk and severity of CKD; (3) these associations remained statistically significant in diabetic and non-diabetic individuals, as well as in studies adjusting for traditional risk factors for CKD, and were independent of whole body/abdominal obesity and insulin resistance.The prevalence of CKD is rapidly growing and in the United States over 1.1 million individuals are estimated to have ESRD by the year 2015 NAFLD is an emerging risk factor for end-stage liver disease and CVD: the frequency of NASH as the primary indication for liver transplantation has increased from 1.2% to 9.7% in the last decade, becoming the third most common indication for liver transplantation in the United States Current guidelines do not recommend screening for CKD in the absence of traditional risk factors for CKD From a therapeutic standpoint, there is a considerable potential for improving the current care of NAFLD patients with CKD: with respect to lifestyle interventions, smoking cessation should be more vigorously pursued, as cigarette smoking is an established risk factor for CKD, and may also aggravate NAFLD Further research is required to unravel the specific cascades linking NAFLD and kidney disease. NAFLD and CKD share common risk factors and therefore both liver and kidney injury may be driven by obesity-associated mechanisms of disease, including lipotoxicity, oxidative stress, enhanced pro-inflammatory cytokine, and renin-angiotensin-aldosterone system (RAAS) axis activation We also found a cross-sectional association between NAFLD and CKD, implying that even mild renal dysfunction may promote liver disease, in a mutual negative loop with detrimental cardio-metabolic consequences Consistently, uni-nephrectomized rats developed body fat redistribution from adipose depots to non-adipose tissues, profound dysregulation of hepatic fatty acid metabolism, steatohepatitis, insulin resistance, hyperglycaemia, and dyslipidaemia early after uni-nephrectomy and long before glomerulosclerosis and chronic renal failure occur Lastly, since NAFLD is an emerging risk factor for liver-related and cardiovascular complications, the hypothesis that the presence of CKD may represent a simple, cost-effective tool to predict increased liver-related and CVD risk in NAFLD is intriguing and warrants assessment in large-scale prospective studies. Currently, in fact, there is no validated non-invasive marker to predict the risk of both liver disease progression and future CVD, the main causes of death in NAFLD patients Our findings may also have therapeutic research implications. Future trials will need to evaluate the impact of experimental treatments on kidney-related outcomes. Notably, only two of the available randomized controlled trials in NAFLD reports the effect of drugs on eGFR and proteinuria and none has adequate size and duration to evaluate the impact of treatments on kidney-related clinical outcomes.Our analysis has limitations, which are intrinsic to the nature of included studies and provide the basis for future research. Studies with biopsy-proven NAFLD were by their own nature less numerous and smaller than those adopting ultrasonographic/biochemical definitions of NAFLD, leaving the possibility of small study bias that is not detected by current tests. Furthermore, these studies were performed in tertiary centers with the possibility of selection bias. Conversely, ultrasound/liver enzyme elevations are relatively insensitive to detect NAFLD, with possible misclassification of individuals with NASH/advanced fibrosis as healthy controls and underestimation of the strength of the association between NAFLD and CKD. However, there was no heterogeneity between studies: disease effect on CKD.Finally, despite our best efforts IPD were unavailable from as much as 39% of relevant studies, representing 54% of participant population. While meta-analysis of AD has several limitations, including ecological bias and study-level confounding, excluding such a substantial proportion of relevant literature from our analysis would have raised the concern of data availability bias Balancing all these reasons, we presented all relevant literature evidence by combining IPD with AD in the main analysis, and separately analyzed studies providing IPD with a one-stage method: notably, the two analyses yielded similar results, further supporting the robustness of overall findings.In conclusion, our analysis shows that the presence and severity of NAFLD are associated with an increased risk and severity of CKD and may be a target for the prevention and treatment of CKD. Future research should evaluate strategies and interventions to prevent renal disease progression in individuals with NAFLD.Text S1PRISMA checklist.(PDF)Click here for additional data file.Text S2Search strategies and protocol.(PDF)Click here for additional data file.Text S3Additional analyses.(DOC)Click here for additional data file.


Background and aims. The aim of the present study is to determine the incidence of MTHFR C677 T and A1298C muta-tions in Iranian patients with cleft lip and/or cleft palate.Materials and methods. We screened 61 Iranian patients with cleft lip and/or cleft palate for mutations in the two alleles of MTHFR gene associated with cleft lip and/or palate: A1298C and C677T, using Polymerase Chain Reaction following by RFLP.Results. The 677T and 1298C homozygote genotypes showed a frequency of 36.1% and 11.4%, respectively. Combined genotype frequencies in newborns having oral clefts showed that the highest genotype was 677TT/1298AA (22.9%) and 677TT/1298CC genotypes were not observed.Conclusion. The results showed that 65.6% of all patients had at least one T mutant allele in C677T and 58.9% C mutant allele for A1298C. According to the frequencies of homozygosity of mutant alleles, it could be said that MTHFR genotype of 677TT shows a greater role in having oral clefts. This gene translates to an enzyme which catalyzes the methylation of homocysteine amino acid to methionine. Defects in this pathway lead to metionine deficiency and the accumulation of homocysteine.Evidence shows that variations in genes associated with folate metabolism may greatly take part in the etiology of non-syndromic CL/P. Among genes related to folate metabolism, the methyletetrahydrofolate reductase gene (9 Two common polymorphisms (C→T and A→C) exist within the MTHFR gene at positions 677 and 1298, respectively.10 Results regarding the association of MTHFR gene polymorphism and the risk of nonsyndromic CL/P have been contradictory. None of the previous studies have examined Iranian patients. Therefore, the aim of this study was to assess the role of MTHFR C677T and A1298C in the development of non-syndromic CL/P in an Iranian population.Despite the critical role of methionine as an important precursor in the DNA and RNA methylation process, high serum homocysteine levels are teratogenic during the embryogenesis.Sixty-one patients with CL/P from those who were referred to Mofid Hospital in Tehran, Iran, were selected for the purpose of this study. The selected patients had non-syndromic cleft lip and/or palate; those with other facial or skeletal malformations, metabolic or neurologic disorders or anomalies of other organs were excluded in case selection.Ethical approval for the study was obtained from the Ethics Committee of the Dental Faculty of the Shahid Beheshti University of Medical Sciences. Written informed consents were obtained from all participants. In order to identify any possible prenatal contributory teratogenic factor that might have influenced the development of CL/P, a detailed questionnaire was completed. The questionnaire was modeled on the United States Centers for Disease Control (CDC) questionnaire for risk factor surveillance for birth defects. We asked all mothers for maternal illnesses, medication intake, history of abortion, history of cardiovascular disease, smoking and amount of folate intake during the periconceptional period (ranging from 3 months prior to 1 month after conception).MTHFR gene mutations was performed by enzymatic restriction digestion of PCR products with HinfI and MboII , respectively.12Blood samples were collected using a sterile 2-ml disposable syringe. Blood samples were collected in tubes containing 200 µl 0.5-M EDTA and stored at −80°C until further analysis. Genotyping for C677T and A1298C MTHFR gene, exon 4 and 7 of the gene were amplified by polymerase chain reaction (PCR) following standard conditions and with the use of modified primers: . DNA fragments were separated and visualized by electrophoresis using 2% agarose gels.For screening of the 677C→T and 1298A→C variants in the Sixty-one subjects were available for analysis. All patients had Iranian background.Tables MTHFR SNP was detected in 36.1%. The frequency of the C677T heterozygotes was 29.5%. Also, homozygosity for the MTHFR A1298C SNP was detected in 11.4% of the tested individuals. 47.5% were heterozygote for this SNP.Homozygosity for the C677T MTHFR has been widely investigated in order to establish its potential role as a risk factor or marker of cardiovascular disease, neural tube defects, cognitive disorders, and cancer,14 despite the frequently conflicting literature.Over the last few years, impaired MTHFR C677T transition induces an enzyme theromlability with an in-vitro reduced enzyme activity ranging from 30-70% in hetero and homozygotes, respectively.15 The MTHFR A1298C polymorphism has been less investigated; the 1298C allele is responsible for reduced enzyme activity although it is less deteriorating than the 677T allele. The phenotypic expression of these mutations, especially for the 677T polymorphism can be conditioned by environmental interactions. In this context, folate is of great importance because an inadequate folate intake in addition to reduced MTHFR activity could facilitate the clinical expression of the MTHFR mutation-disease association.16The 17 heterozygote (CT) and variant homozygote (TT) frequencies of 677TC were 18%– 46%–45% and 0.9%–21%–11%, respectively, for African-American–Hispanics–Caucasians. Furthermore, 677TT variant homozygote in Hispanic newborn population was higher than other ethnic groups which can explain the higher prevalence of neural tube defects in this population.17 Another study in Italy showed the prevalence of 677TT and 1298CC genotypes was 25% and 47.1%, respectively.18The genotype prevalence and allele frequencies of the two polymorphisms have not been reported in CL/P patients previously. In a study conducted by Yanamandra et al,To date, only a few studies have evaluated the A1298C genotype prevalence all over the world. Thus, the results must be confirmed by further larger epidemiologic studies. Several studies describe the absence of other combined genotypes such as the 677CT/1298CC and 677TT/1298AC, which we found at low percentages .MTHFR activity of about 50% and an increase in homocysteine level, the data do not relate to children with oral clefts. Therefore, it could be claimed that TT genotype is an important factor in developing oral clefts, while polymorphism in 1298AC does not have a critical role.1Despite the concern for double heterozygosity of the 677T and 1298C allele, our results are not in agreement with previous studies. Although this genotype combination has been proposed to predispose a reduction in MHTFR genotypes in a newborn population.21 Moreover, the present study evaluated the prevalence of the two MTHFR polymorphisms in an Iranian population with CL/P.The present investigation is among the few studies in this regard that have analyzed the prevalence of MTHFR polymorphisms, folate serum levels in mothers, and the risk of orofacial clefts in children raises the question about the use of dietary supplements containing folic acid by pregnant women. The study of the prevalence of these polymorphisms in different geographical areas could be of interest in evaluating the possible clinical application deriving from the preventive folate supplementation.The close relationship between The authors would like to thank Dr. Roozrokh, Head of Mofid Hospital, the staff at the hospital, and the Genetic Research Center for their kind help in recruiting study subjects. This article was derived from the research performed by Dr. Nazila Ameli for her Post-graduate Degree in Orthodontics under the supervision of Dr. Ebadifar and supported by grants from Dentofacial Deformities Research Center, and Research Institute of Dental Sciences, both at Shahid Behehsti University of Medical Sciences, Tehran, Iran.


Background. The calcium-sensing receptor gene (CaSR) is a candidate to explain urolithiasis. A number of case-control studies were conducted to investigate associations between CaSR polymorphisms with risks of hypercalciuria and urolithiasis in humans. But the results were still inconsistent. Methods. A meta-analysis was performed to address this issue. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to estimate the strength of associations between CaSR polymorphisms and the risk of urolithiasis. The pooled standardized mean difference (SMD) with 95% CI was used for the meta-analysis of CaSR polymorphisms and urine calcium concentration. Results. For urolithiasis association, the SS genotype of A986S polymorphism was a risk factor for urolithiasis in Asians and PHPT patients, but a protective factor in Caucasians. The GG genotype of R990 polymorphism was associated with an increased risk of urolithiasis, especially in Caucasians and healthy population. Regarding urine calcium concentration association, individuals with the G allele had a higher level of urine calcium than the noncarriers. Conclusions. This meta-analysis revealed that the G allele of CaSR R990G polymorphism increases susceptibility to urolithiasis and hypercalciuria. The A986S and Q1011E polymorphisms were associated with urolithiasis and hypercalciuria in specific populations. Urolithiasis is a condition that has been recognized for centuries and is the third most common cause of urinary tract disease . Nearly Urolithiasis is a multifactorial disease which is considered to be associated with the effects of multiple genes in combination with lifestyles and environmental influences . AlthougThe CaSR gene is composed of seven exons, the first six coding for the extracellular domain of the CaSR protein and exon 7 coding for the transmembrane and the intracellular domains . Three sAt present, several studies have attempted to investigate associations between CaSR gene variants with urolithiasis and urinary calcium concentration. However, the results were inconsistent or even contradictory. To date, no one has conducted a meta-analysis to further probe the associations. To fill this gap, we performed a meta-analysis of all eligible studies to derive more reliable estimation of associations between calcium-sensing receptor gene polymorphisms with urolithiasis and urinary calcium concentration.A comprehensive literature search was performed through the PubMed, Medline, Embase, and Web of Science databases for relevant articles published  with the following key words: “CaSR,” “polymorphism,” “variation,” or “mutation,” and “urolithiasis,” or “calculi” and in combination with “urine calcium excretion.” Additional studies were identified by hand, searching references in original articles and review articles.The included studies needed to meet the following criteria. (1) The study examined the associations between CaSR polymorphisms and urinary calcium concentration and/or urolithiasis risk. (2) For urolithiasis association, the study must be case-control study and must have clear original data of genotypic and allelic frequencies. (3) For urinary calcium concentration association, the study must have clear original data of the mean of urinary calcium concentration and standard deviations (SD) by genotypes. In addition, the number of each genotype must be clear. Major reasons for exclusion of studies were as follows: (1) not for urolithiasis or urinary calcium concentration research, (2) review articles, (3) only case population, and (4) duplicate of previous publication.Information was carefully extracted from all eligible studies independently by two investigators according to the inclusion criteria listed above. For conflicting evaluation, a consensus was reached by discussion. The following information was collected from each study: the first author's name, the year of publication, ethnicity, country of origin, genotyping method, source of control groups , subjects, numbers of cases and controls, frequency of CaSR polymorphisms in cases and controls, the mean of urinary calcium concentration, and SD by genotypes. Meanwhile, the corresponding and first authors of the published studies were contacted by sending e-mails if they did not provide their original data.Q test  [P > 0.10 for the Q test). Otherwise, the random-effects model would be applied. To better investigate the possible sources of between-study heterogeneity, meta-regression analysis was performed. In addition to the comparison among all subjects, we also conducted stratification analyses by ethnicity, subjects, and source of controls. Sensitivity analyses were performed to assess the stability of the results; namely, a single study in the meta-analysis was deleted each time to reflect the influence of the individual data set to the pooled OR. The pooled standardized mean difference (SMD) with 95% CI was used for the meta-analysis of CaSR polymorphisms and urinary calcium concentration. Begg's funnel plot and Egger's linear regression test were used to assess publication bias. Moreover, departure from Hardy-Weinberg equilibrium (HWE) in controls was tested by the chi-square test for goodness of fit, and a P < 0.05 was considered as a significant disequilibrium. All statistical analyses were performed with the Stata software , using two-sided P values.Crude odds ratios (ORs) with their corresponding 95% CIs were used to assess the strength of associations between CaSR polymorphisms and urolithiasis risk. The pooled ORs were performed for homozygote model (M/M versus W/W), heterozygote model (W/M versus W/W), dominant model (W/M + M/M versus W/W), and recessive model (M/M versus W/W + W/M), respectively. Between-study heterogeneity was checked by the chi-square-based  < 0.10) . Pooled There were 43 articles relevant to the search words, of which 34 articles were excluded. Of the 34 excluded studies, 11 articles were not related to A986S, R990G, and Q1011E polymorphisms, 8 studies were review articles, and 15 papers were not case-control studies. Besides, 3 additional articles , 20, 21 For the associations between CaSR polymorphisms and urolithiasis, a total of seven papers which included 10 case-control studies , 13–18 fFor the associations between CaSR polymorphisms and urinary calcium concentration, ten papers , 18–23 wThe relationship between the R990G polymorphism and the risk of urolithiasis is summarized in As shown in P = 0.039; P = 0.56) and the Q1011E polymorphism . Furthermore, we performed subgroup analysis (P = 0.026). Unfortunately, we did not obtain other positive findings.The results of the overall meta-analysis provided a strong evidence of the association between urinary calcium concentration and the CaSR R990G polymorphism .There was heterogeneity among studies in overall comparisons. To explore the sources of heterogeneity, we conducted subgroup analyses by ethnicity and subjects. However, the heterogeneity remained significant. Egger's test and Begg's funnel plot were applied for comparison to assess the publication bias of the literature, and no possibility of publication bias for this test was observed  wasting and hyperphosphaturia and it perhaps resulted from their significant polyuria and urinary acidification. Activation of the renal CaSR promoted H+-ATPase-mediated H+ excretion and downregulation of aquaporin 2 (AQP 2), leading to urinary acidification and polyuria, respectively. The mice developed calcium-phosphate precipitate in collecting ducts only after inhibition of H+-ATPase activity that hampers urine acidification. Thus, we thought that CaSR polymorphisms might be involved in urolithiasis via influencing activities of CaSR gene and H-pump.A growing body of evidence shows that the CaSR might play a crucial role in the pathogenesis of urolithiasis. CaSR is a common protein which usually expressed in the parathyroid glands, renal tubules, and distal tubules . CaSR isThe incidence of gene polymorphisms can vary substantially among different racial populations. We therefore performed stratified analysis by ethnicity. Interestingly, contradictory associations were found in the A986S and R990G polymorphisms. For the A986S polymorphism, the SS genotype was associated with an increased urolithiasis risk in Asians but a decreased risk in Caucasians. Regarding the R990G polymorphism, the association between GG genotype and an increased risk of urolithiasis was only found in Caucasians. Even though the exact mechanism for the results was not well known, some concerns may account for it. Firstly, we presumed that the difference among ethnic groups might be a reflection of different genetic backgrounds and environmental context. Secondly, the sample size was relatively small, not having enough statistical power to explore the real association. Moreover, in view of diversity of possible comparisons and unavoidable flexibility of defining the correlations, associations may not necessarily reliable. For instance, selection bias, different matching criteria may play a role.Different research objects may have an influence on the conclusions. We also conducted stratified analysis by subjects. Different subjects were categorized as PHPT patients, healthy population, and mixed population. Primary hyperparathyroidism is a disease characterized by excessive parathyroid cell proliferation and PTH secretion and occurs frequently in postmenopausal women , 35. KidIt is worth noting that hypercalciuria, a disorder predisposing to calcium kidney stones, is vital in the mechanism of urolithiasis; therefore, we performed the first meta-analysis to evaluate the relationships between three CaSR polymorphisms and urine calcium concentration. The calcium-sensing receptor is the key controller of extracellular calcium homeostasis via its effects on regulation of parathyroid hormone secretion and renal calcium reabsorption . HypercaFurthermore, despite the overall robust statistical evidence generated through this analysis, some limitations have been identified. Firstly, our results were based on unadjusted estimates, while a more precise analysis should be conducted if all individual raw data were available, which would allow for the adjustment by other covariates including age, sex, family history, and lifestyle. Secondly, only published studies which were retrievable in the selected databases were included in this meta-analysis, and some unpublished studies were missed. Thirdly, urolithiasis is a multifactorial disease that results from complex interactions between many genetic and environmental factors. It suggests that there will not be single gene or single environmental factor that has large effects on urolithiasis susceptibility. Fourth, heterogeneity could not be omitted because of methodological diversities between studies. Last but not least, this is the first meta-analysis regarding the comprehensive assessment of the relationship between CaSR polymorphisms with urolithiasis risk and urine calcium concentration. So numbers of published studies were not sufficiently large for a comprehensive analysis. The populations only come from Asians and Caucasians. Other ethnic populations should be involved in the future studies, such as Africans. Only four papers evaluated associations between the Q1011E polymorphism and urolithiasis risk; more studies should be conducted.Despite these limitations, the results of the present meta-analysis suggest that the G allele of CaSR R990G polymorphism increases susceptibility to urolithiasis and hypercalciuria. In other words, individuals that carry GG genotype have a higher risk of urolithiasis than those who carry RR genotype. The A986S and Q1011E polymorphisms were associated with urolithiasis and hypercalciuria in specific populations.The identification of urolithiasis susceptible variants can provide new insight into its etiology. Moreover, it is an important step to individualize treatment and prevention programs. A well-established genetic marker surely would have a profound influence in screening and prediction of urolithiasis. To advance an understanding of the relationships between CaSR polymorphisms with urolithiasis risk and hypercalciuria, the following recommendations have been made. Firstly, try to decrease false positive and negative results by conducting the studies in a large sample with stratification by age, sex, food habit, lifestyle, and ethnicity. Secondly, more case-control studies or updated meta-analyses should be conducted to clarify the possible roles of CaSR polymorphisms in the etiology of urolithiasis. In addition, because the genetic background of stone formation is a complicated issue including single-candidate genes as well as epigenetic process, it is not simple to identify a single gene as an independent factor for urolithiasis. Combined effects of different gene polymorphisms need to be further analyzed.


CHRNA3 gene and lung cancer risk has been studied in Chinese by several number case-control control studies with small number of cases and controls, and these studies might be underpowered to reveal the true association. Thus we sought to investigate the association with the risk of lung cancer by performing a comprehensive meta-analysis on the polymorphism. Five case-control studies were extracted from 3 articles on the polymorphism involving 4608 lung cancer cases and 4617 controls. The results of meta-analysis showed that significant increased risk were found for the polymorphism with the risk of lung cancer in Chinese: OR = 1.47, 95%CI = 1.33–1.63, P < 0.00001 for CC + TC vs. TT; OR = 1.24, 95%CI = 1.07–1.44, P = 0.005 for CC vs. TT + TC; OR = 1.62, 95%CI = 1.32–2.00, P < 0.00001 for CC vs. TT; OR = 1.42, 95%CI = 1.26–1.61, P < 0.00001 for CT vs. TT; OR = 1.42, 95%CI = 1.26–1.61, P < 0.00001. No significant publication bias was found for the five genetic models. Our findings demonstrated that CHRNA3 gene rs6495309 polymorphism might be a risk factor for the development of lung cancer in Chinese.The association between the rs6495309 polymorphism in  CHRNA3 polymorphisms8Lung cancer is one of the most common malignant tumors in humans and is the most common cause of cancer-related mortality135CHRNA3 gene is located on the chromosome 15q25.1 region which has been identified as a hotspot for lung cancer susceptibility by recent genome-wide association (GWA) studies101110111314The human th, 2014) to identify all relevant publications on the association between CHRNA3 rs6495309 polymorphism and lung cancer risk in Chinese. The following search terms and their synonyms were used: lung cancer and polymorphism and CHRNA3 and Chinese. We also manually searched the reference lists of all eligible studies and review articles to obtain additional usable data that can be included in the current meta-analysis.The databases of PubMed, Embase, CNKI and Wanfang were searched  adequate genotyping data must be contained such that odds ratios (ORs) with 95% confidence intervals (CIs) could be calculated; (4) the study had to be published using human subjects. Exclusion criteria were: (1) insufficient information on the distribution of CHRNA3 genotypes; (2) case-only studies; (3) duplicated publications. If a study was subsequently updated, we selected the study with the largest sample size. Two investigators independently reviewed all studies to examine whether they fulfilled the inclusion criteria.We selected eligible studies according to the following criteria: (1) the study must have a case-control design; (2) the association between Two independent investigators extracted the original data according to the inclusion criteria and exclusion criteria to ensure the accuracy of the retrieved information. The data extracted from each eligible study included the first author's name, year of publication, cancer type, ethnicity, source of controls, method adopted for genotyping, number of cases and controls and genotype frequencies. Disputes were settled by consulting the third person.2I statistics, P > 0.05 for the Q test or 2I < 50% suggested a lack of heterogeneity. In this situation, the OR of each study was calculated by the fixed-effects model (the Mantel-Haenszel method). If P < 0.05 or 2I > 50%, the random-effects model (the DerSimonian and Laird method) was used16P < 0.05 was accepted as statistically significant.Hardy-Weinberg equilibrium (HWE) of the control groups was tested by the χ2 test for goodness of fitness. Crude ORs with 95% CIs were calculated to evaluate the strength of the association between the polymorphism and lung cancer risk. The pooled ORs were performed for the following genetic models: allele contrast (C vs. T), homozygote (CC vs. TT), heterozygote (CT vs. TT), dominant (CC + CT vs. CC) and recessive (TT vs. CT + CC) model. Heterogeneity assumption was evaluated by the chi-square based Q-test and The screening of the studies is shown in 191920CHRNA3 rs6495309 polymorphism and lung cancer risk for Chinese . Begg's funnel plots and Egger's test were performed to evaluate publication bias in the literature. Funnel plots of the genetic model seemed symmetrical . This was confirmed by the statistical data derived using Egger's test .The five case–control studies included in the quantitative analysis yielded a total of 4608 lung cancer cases and 4617 controls for the CC + CT vs. TT comparative . No signCHRNA3 rs6495309 polymorphism and lung cancer risk for Chinese . Begg's funnel plots and Egger's test were performed to evaluate publication bias in the literature. Funnel plots of the genetic model seemed symmetrical . This was confirmed by the statistical data derived using Egger's test .The five case–control studies included in the quantitative analysis yielded a total of 4608 lung cancer cases and 4617 controls for the CC vs. TT + CT comparative . SignifiCHRNA3 rs6495309 polymorphism and lung cancer risk for Chinese . Begg's funnel plots and Egger's test were performed to evaluate publication bias in the literature. Funnel plots of the genetic model seemed symmetrical. This was confirmed by the statistical data derived using Egger's test .The five case–control studies included in the quantitative analysis yielded a total of 2262 lung cancer cases and 2369 controls for the CC vs. TT comparative . SignifiCHRNA3 rs6495309 polymorphism and lung cancer risk for Chinese . Begg's funnel plots and Egger's test were performed to evaluate publication bias in the literature. Funnel plots of the genetic model seemed symmetrical. This was confirmed by the statistical data derived using Egger's test .The five case–control studies included in the quantitative analysis yielded a total of 3106 lung cancer cases and 3290 controls for the CT vs. TT comparative . No signCHRNA3 rs6495309 polymorphism and lung cancer risk for Chinese . Begg's funnel plots and Egger's test were performed to evaluate publication bias in the literature. Funnel plots of the genetic model seemed symmetrical. This was confirmed by the statistical data derived using Egger's test .The five case–control studies included in the quantitative analysis yielded a total of 4608 lung cancer cases and 4617 controls for the C vs. T comparative . SignifiCHRNA3 gene with the risk of lung cancer for Chinese. Although potential sources of heterogeneity could not be easily eliminated, the present study, to our knowledge, is the first meta-analysis to date dealing with the association of the rs6495309 polymorphism with lung cancer susceptibility for Chinese.Via a comprehensive meta-analysis, we evaluated the association of one common polymorphism in the CHRNA3 rs6495309 polymorphism and lung cancer in Chinese under all models: allele contrast (C vs. T), homozygote (CC vs. TT), heterozygote (CT vs. TT), dominant (CC + CT vs. CC) and recessive (TT vs. CT + CC) model. Thus, the CHRNA3 rs6495309 polymorphism could be suggested as a lung cancer risk factor for Chinese.In this meta-analysis, we included a total of five case-control studies. The pooled results indicated that there were obvious associations between vs. TT is the largest among all five ORs; thus, the individuals who carries the CC homozygote might have more increased risks. Previously, Wu et al. reportedAlthough previous case-control studies suggested the rs6495309 polymorphism might contribute to the risk of lung cancer for Chinese, however, these studies were with small number of cases and controls. Thus, we performed the current meta-analysis. In the current meta-analysis, the results showed that the value of the OR for CC Meta-analysis is a useful method for investigating associations of diseases with genetic factors because it uses a quantitative approach by way of combing the results of different studies on the same topic, and potentially providing more conclusive results242526CHRNA3 polymorphism and lung riskCHRNA3 rs1051730 polymorphism and lung cancer was acquired. Compared with the previous meta-analysis, we added another CHRNA3 polymorphism that might contribute lung cancer risk in Chinese using newly published studies.As far as we know, there has been one published meta-analyses regarding CHRNA3 rs6495309 polymorphism and the risk of lung cancer, further investigations with larger sample sizes and higher quality are needed. Third, the overall outcomes were based on individual unadjusted ORs; a more precise estimation should be adjusted by menstrual status, age, environmental and other confounding factors. Finally, this meta-analysis could not address the gene-gene and gene-environmental interactions in the association between CHRNA3 rs6495309 polymorphism and lung cancer risk. Future studies that include detailed information on exposures to various carcinogens and individual-level data to assess the possible gene-gene and gene-environment interactions in the association between CHRNA3 rs6495309 polymorphism and lung cancer risk are needed.Several limitations should be acknowledged when interpreting the results of this meta-analysis. First, there was a potential language bias, because the PubMed, EMbase, CNKI and Wanfang search engines were used to identify articles and to exclude articles written in languages other than English and Chinese. This might not have prevented the researchers from accessing all relevant studies. Second, relatively few eligible studies, all with small sample sizes, were included in this meta-analysis, which could increase the risk of random error. To conduct a more precise analysis of CHRNA3 rs6495309 polymorphism is associated with an increased risk of lung cancer in Chinese. Additional studies with a greater number of patients should be performed to examine how the CHRNA3 variants interact with other risk loci to influence lung cancer risk.This meta-analysis of five case-control studies suggested that F.W. designed the study, wrote the manuscript. M.X., L.C. and X.W. performed the analyses. All authors reviewed the manuscript.


S-transferases (GSTs) are enzymes which expressed in many tissues and play important roles in neutralization of toxic compounds, and protecting hosts against cancer. Among several GSTs, Glutathione S-transferases mu (GSTM) has been drawn attention upon the association with the genetic risk for many types of cancers. But whether the GSTM1 polymorphisms confer the susceptibility to colorectal cancer in Asians has not been well established. We searched the PubMed database with GSTM1, polymorphism and colorectal cancer, attempting to identify the eligible studies. In total, 33 case-control studies in Asian populations with 8502 colorectal cancer patients and 13699 controls were included in the current meta-analysis. The association between the polymorphism and susceptibility to colorectal cancer was evaluated by the odds ratio (OR) and 95% confidence intervals (CI). The pooled meta-analysis suggested that GSTM1 null variant was correlated to the colorectal cancer risk in Asians. There was a marginal heterogeneity among these eligible studies. Nevertheless, cumulative meta-analysis observed a trend of an obvious association between the GSTM1 null genotype and colorectal cancer risk in Asians. In summary, the meta-analysis suggested that GSTM1 null polymorphism confer the susceptibility to colorectal cancer in Asians, especially in Chinese populations.Glutathione  Colorectal cancer is the second leading common cancer and a major cause of cancer-related deaths in the world-wide, which accounts for about 9.7% death rate of all cancers135S-transferase M1 (GSTM1) encodes the mu class of GSTs, which plays vital roles in protecting hosts against cancersGSTM1*0 allele) is the most common variant of the GSTM1, leading to the loss of enzyme activity, and the variant-carriers were proven to be associated with increased risk to cancersGSTM1 has been identified to be involved in the pathogenesis and development of certain cancers, inclusive of colorectal cancerGSTs are a super-family of phase II detoxification enzymes which play critical roles in the detoxification of exogenous and endogenous reactive species through conversion of toxic compounds to hydrophilic metabolites9GSTM1 null variant was correlated with the risk of cancers15GSTM1 null genotype has been demonstrated to be linked to early onset for colorectal cancerGSTM1 polymorphism and the colorectal cancer susceptibility in Chinese populations were controversial, and so were the genetic results in Asians. Inclusive of 33 case-control studies, the present meta-analysis was performed to further explore the relationship between GSTM1 null variant and the susceptibility to colorectal cancer more comprehensively in Asians.Many association studies and evidence were conducted and highlighted the http://www.ncbi.nlm.nih.gov/pubmed/) to identify the eligible case-control studies using the following keywords: (“glutathione S-transferase” or “GST” or “glutathione S-transferase M1” or “GSTM1”), (“polymorphism” or “Single Nucleotide Polymorphism”) and . There was no language limitation in the literature search. Titles and abstracts of these searching studies were primarily screened and full papers were further retrieved to confirm eligibility, the reference lists were also examined to find other relevant studies. These studies were reported from 1996 to 2014. These given studies included into our meta-analysis had to meet the following inclusion criteria: (1) case-control validation study, (2) the studies should estimate the association of the GSTM1 null variant with risk to colorectal cancer, (3) the studies should provide odds ratio (OR) with 95% CI or available data, (4) the studies should be conducted in Asian populations. Reviews and duplicate studies were excluded from the analysis. If the same case-control study were overlapped in multiple publications, only the most complete or most recent literature was included in the present study.We systematically searched the PubMed database  or absence (I2 > 50%) of significant heterogeneity. So, the random-effect model should be chosen once the obvious heterogeneity was observed. Interesting, the heterogeneity was also considered to be significant when P < 0.101821P < 0.05 in the meta-analysis2426P values were two-sided.The association between The flow chart of eligible studies was shown in P = 0. 01, I2 = 40.4%)  in a fixed-effect model   . Overallct model . The ass02–1.11)  under th02–1.11) .GSTM1 null genotype and colorectal cancer risk in Asians as information accumulated by year  was used, the publication bias was more significant in the Egger’s regression test   . Nine mi < 0.05) . The comP = 0. 023, I2 = 42%)There were 21 studies of Chinese, 6 in Japanese, 2 of Taiwan subjects, each single study in Korean, Singapore, India and Iran in the meta-analysis. Sub-analysis based on the different countries was conducted to verify the heterogeneity, it seemed that there may exist a marginal heterogeneity in Chinese populations . Third, evident bias also exists accordingly to the publication bias analyses. More case-control studies should be added to conduct the meta-analysis, then to further detect the potential gene-gene and gene-environment association.Compared to the previous meta-analyses, more validation studies were pooled in the present meta-analysis. The pooled meta-analysis, the cumulative meta-analyses by publication year and the sample size, and the sensitivity analysis correspondingly supported the GSTM1 null variant was significantly associated with the susceptibility to colorectal cancer in Asians, which supporting the genetic factors play vital roles in the pathogenesis of colorectal cancer. Further validation studies should be included to solidify the current conclusions.In summary, the meta-analyses implied that the How to cite this article: Li, J. et al. GSTM1 polymorphism contribute to colorectal cancer in Asian populations: a prospective meta-analysis. Sci. Rep.5, 12514; doi: 10.1038/srep12514 (2015).