Purpose. To clarify the association of endothelial nitric oxide synthase (eNOS) polymorphisms and primary open angle glaucoma (POAG). Methods. After a systematic literature search in the MEDLINE, EMBASE, and ISI Web of Science databases, all relevant studies evaluating the association between the polymorphisms (rs2070744 and rs1799983) of eNOS gene and POAG were screened and included. The pooled odds ratios (ORs) and the 95% confidence interval (CI) of each single-nucleotide polymorphism (SNP) in five genetic models were estimated using fixed-effect model if I2 < 50% in the test for heterogeneity; otherwise the random-effects model was used. Results. Thirty-one records were obtained, with five being suitable for meta-analysis. The overall results showed that both TT genotype in rs2070744 and GG genotype in rs1799983 are associated with decreased risk of POAG susceptibility. Stratified analysis based on ethnicity showed that the association of rs2070744 with POAG remained only in Caucasians. Results of subgroup analysis by sex indicated association between both polymorphisms and POAG in female group, but not in male group. Conclusions. TT genotype and/or T-allele in rs2070744, as well as GG genotype and/or G-allele in rs1799983, was associated with decreased risk for POAG overall and in female group. Glaucoma is a common, complex, heterogenous disease and it constitutes the major cause of irreversible blindness worldwide . In 2013Nitric oxide (NO) is an active biologic agent involved in diverse physiologic processes . NO geneA systematic literature search was conducted in the MEDLINE, EMBASE, and Web of Science databases  with the following free words and MeSH terms: “glaucoma”, “open angle”, “Endothelial nitric oxide synthase”, “eNOS”, “polymorphism(s)”, “single nucleotide polymorphism”, and “SNP”. We also supplemented our search by screening the reference lists of all the retrieved studies, as well as genome-wide association studies (GWAS) performed for glaucoma to which we have the access.Eligible studies were included if they (1) evaluated the association between eNOS and POAG; (2) compared unrelated POAG cases and normal controls identified by complete ophthalmological examination in defined populations; (3) provided an odds ratio (OR) with 95% confidence interval (CI) in case and control groups, respectively, or other data which could be calculated to estimate an OR; and (4) were original research articles. All animal studies, case reports, abstracts from conferences, full-texts with incomplete data, and reviews were excluded. As for duplicate studies retrieving data from the same source, ones with available data and the largest sample size were brought into the analysis list. Although we did not define language in the review process, the articles in the final analysis were all in English.Two investigators (Yang Xiang and Yi Dong) extracted data from the retrieved records and confirmed the validity of the included articles independently. The following variables were extracted: author, year of publication, ethnicity of subjects, demographic information, the numbers of cases and controls, results of Hardy-Weinberg equilibrium (HWE) test when reported, and the allele and genotype counts or frequencies of each SNP. When the allele or genotype counts were not given specially in some articles, they were calculated from the frequencies and then rounded to the nearest integer. A final review was performed by other reviewers (Xuan Li and Xin Tang) while the discrepancy was resolved through discussion.The Chi-square test was utilized to check whether the genotype distribution in controls was consistent with HWE for studies that did not report relevant data. To assess the strength of association between the polymorphisms (rs2070744 and rs1799983) of eNOS gene and POAG susceptibility, we estimated crude OR with its 95% CI under allele model , homozygote model , heterozygote model , dominant model , and recessive model , respectively.Q statistic test as well as the I2 metric. If PQ ≤ 0.10 or I2 > 50%, which indicated significant heterogeneity in the comparison models among studies [P < 0.05 were considered statistically significant.Interstudy heterogeneity was detected using the Chi-square-based  studies , the poo studies . Otherwi studies . We also studies , 25. TheThe workflow and results of the literature review are shown in Tables To further explore the association, stratified analysis was performed based on ethnicity (Caucasians and Asians) and sex. For rs2070744, the results showed that the association between rs2070744 and POAG was significant in Caucasians  but not in Asians . StratifPublication bias was quantitatively assessed by Begg's tests and Egger's tests. In the overall analysis, there was no evidence of publication bias detected for rs2070744 . With reWe reviewed a broad selection of publications found in electronic databases and performed a meta-analysis, in an attempt to identify the effects of polymorphisms of the eNOS gene on the pathogenesis of POAG. Five eligible studies were involved and available data in this regard were conflicting –19. AfteGenerated by eNOS via the conversion of L-arginine to L-citrulline, NO acts as a pivotal vasodilator mediator liberated from endothelial cells of ocular blood vessels. There is evidence that constant formation of NO by eNOS provides the maintenance of a basal vasodilator tone in the optic nerve head of humans and experimental animals –30, whicRs2070744 and rs1799983 are the most important identified functional polymorphisms of the eNOS. The polymorphism of the promoter region of eNOS rs2070744 has been considered to be related to nonarteritic anterior ischemic optic neuropathy (NAION), coronary spasm, myocardial infarction, and coronary artery disease –39. ThisIn our study, we observed associations between eNOS gene variants and POAG, particularly among the women, revealing some sex-related facts in pathogenesis. Several lines of evidence suggest the sexually dimorphic effects of eNOS. In a series of animal studies, the expression levels of eNOS exhibited sex disparity  and dispFor our study, we have put considerable efforts and attempted to minimize every bias and gain stable and reliable results; however, there are still some limitations. Firstly, studies involved in the present meta-analysis were limited to published full-text articles in English. We failed to track the unpublished articles or ones published in other languages to obtain data for analysis, causing an influence on the completeness of the data. Secondly, although we collected and reviewed all the relevant studies, only five eligible ones were included for analysis and the sample size of the individual studies was not sufficiently large, which could increase the likelihood of type I and type II errors. As for rs2070744, we excluded one study with significant HWE deviation, further decreasing the overall sample size of our study. Therefore our results should be interpreted with caution until these findings can be replicated in other large datasets. Stratified analysis of ethnicity and sex also encountered the similar problem due to the lack of detailed data. Despite all of these limitations, we believe our study would be beneficial to a better understanding of the association between eNOS polymorphisms and POAG. Moreover, our analysis has also revealed the limitations in the current POAG genetic studies. Hence, large-scale and well-designed studies are warranted in the future. As stated, glaucoma was estimated to disproportionally affect people in Africa and thus more research needs to be conducted in the African population. Finally, since POAG is a multifactorial disease and the roles of several genes in the pathogenesis of POAG have been established, further investigations should be performed in this direction. It is possible that specific gene-gene and gene-environment interactions may alter those associations between gene polymorphisms and POAG. We expect that as more studies become available, a more accurate estimation of the relationship of eNOS with POAG will be obtained.In summary, the current meta-analysis suggests that TT genotype and/or T-allele in rs2070744, as well as GG genotype and/or G-allele in rs1799983, was associated with decreased risk for POAG overall and in female group. To better understand the role of genetic factors in the physiopathology of this condition, further studies are needed in large, standardized, and ethnically diverse populations.


FokI polymorphism located in the gene encoding the vitamin D receptor (VDR), and inconsistent conclusions had been reached among different ethnic populations. In order to assess the association between the FokI polymorphism and the risk of IDD, we performed a comprehensive and systematic meta-analysis. Candidate articles were retrieved from PubMed, EMBASE, China National Knowledge Infrastructure (CNKI), and China Biology Medical (CBM) with strict inclusion criteria in January 2015. Among the 54 articles that were retrieved, only eight studies met the inclusion criteria. The pooled data analysis based on allele contrast, homozygote, heterozygote, dominant, and recessive models revealed no significant correlation between the FokI polymorphism and the risk of IDD. However, when stratified by ethnicity, significant associations were detected for Hispanics based on allele contrast , homozygote , heterozygote , and dominant  models, and for Asians using the dominant model , whereas there is no significant association detected for Caucasians. In conclusion, FokI polymorphism is not generally associated with IDD, but there is increased risk for IDD in Hispanics and Asians carrying FokI allele T.Intervertebral disk degeneration (IDD) is strongly associated with genetic predisposition and environmental susceptibility. Several studies been conducted to investigate the potential association between IDD and  The intervertebral disk functions as a fibrocartilaginous cushion pad against mechanical load from the coronal, sagittal, and transverse planes. Pathophysiological intervertebral disk degeneration (IDD) is primarily characterized by deterioration and dysfunction of the annulus fibrous and nucleus pulpous, resulting in a range of symptoms such as chronic neck pain, lower back pain, and upper and lower extremity pain VDR gene, located on chromosome 12 (12q12–q14), spans over 105 kb with a remarkably large promoter VDR is now considered to be one of the genetic biomarkers associated with IDD VDR gene polymorphisms, such as TaqI (rs731236), FokI (rs2228570), and ApaI (rs7975232) Recently, several institutions have reported diverse genetic biomarkers related to IDD, including the polymorphisms in the gene encoding vitamin D receptor (VDR) VDR gene, which leads to an alternative translation start site The FokI polymorphism (C to T transition) is located in the start codon of the VDR FokI T/C transition with IDD was reported in Hispanics VDR FokI T/C transition and IDD in Chinese. Furthermore, Xu et al. performed a meta-analysis VDR FokI polymorphism and IDD. In order to assess the association between the FokI polymorphism and the risk of IDD, our meta-analysis systematically and comprehensively evaluated the evidence accumulated to date with relatively larger sample size and higher statistical power.In addition, many studies We retrieved 54 articles through literature search of PubMed, EMBASE, China National Knowledge Infrastructure (CNKI), and China Biology Medical (CBM). After excluding articles such as duplicates and non-case controlled studies , eight studies were finally included in the meta-analysis . Among tVDR FokI polymorphism and the increased susceptibility to IDD in the pooled population. The results were obtained based on different evaluation models, including allele contrast , homozygote , heterozygote , dominant , and recessive  models.Overall, this meta-analysis did not detect any significant correlation between the P = 0.018), homozygote , heterozygote , and dominant  models for Hispanics, and in the dominant model  for Asians. On the other hand, no statistical significance was revealed in Caucasians. The pooled ORs and 95% CI based on the allele contrast, heterozygote, homozygote models dominant and recessive models are listed in When stratified by ethnicity, however, significant associations were detected in the allele contrast , homozygote (P = 0.540), heterozygote (P = 0.223), dominant (P = 0.426), and recessive models (P = 0.154).We next evaluated the sensitivity in this meta-analysis. No matter which article was rejected, the pooled results did not vary remarkably for the allele contrast model  and the VDR gene was downregulated in skeletal muscle of the aged people. More important, VDR gene variation is highly associated with dysfunctions of vitamin D metabolism VDR gene can cause alteration in the structure of the VDR protein The vitamin D endocrine system is involved in a range of biochemical processes A series of studies etc. Additionally, it was difficult to adopt an adjustment model to eliminate these interfering factors efficiently, because there are too many covariates, compared with relatively small sample size. Second, the diagnoses of IDD were not identical, especially when it comes to the different clinical manifestations. Third, only one study Although the current meta-analysis shows prominent strength of more cases and controls, the limitations of this study must also be taken into serious consideration. First, the calculations of pooled ORs combined with 95% CI were based on the original articles, without adjusting for BMI, age, gender, hard physical labor, tobacco consumption, In conclusion, after comprehensively and systematically evaluating the available literature published in Chinese or English, our meta-analysis suggests that allele T at FokI polymorphism site is highly associated with IDD in Hispanics and Asians, but not in Caucasians. Given the aforementioned limitations of this study, further well-designed studies are needed to validate the correlation between the FokI polymorphism and IDD risk and explore the underlying mechanisms.For comprehensive evidence retrieval, we searched the candidate articles in several electronic bibliographical databases, including PubMed, EMBASE, CNKI, and CBM literature databases on January 15, 2015 without any limitation. The keywords for searching are “vitamin D receptor OR VDR” OR FokI, “polymorphisms OR single nucleotide polymorphism OR SNP OR variation”, and “intervertebral disk degeneration OR intervertebral degenerative disease OR IDD”. In order to obtain more studies, we retrieved the reference lists of the selected studies, reviews, and meta-analyses. The literature search was performed by two authors, and any conflict was resolved through discussion.Articles included in this meta-analysis had to meet the following criteria: (1) investigating the correlation between the polymorphism and the risk of IDD, (2) case–control design, (3) providing the exact number or frequency of alleles and genotypes, (4) distribution of genotype frequencies in control groups following HWE, and (5) publication in English or Chinese, and (6) having the larger (or largest) sample size if more than one papers published are based on the same population. Exclusion criteria include (1) reviews or meta-analysis or non-case-controlled design, (2) the exact number or frequency of alleles and genotypes not available, (3) genotype frequencies in controls not following HWE, and (4) duplicated reports based on the same population.After eligible articles were identified, data were extracted by two independent authors. The following information was extracted from the articles: name of the first author, year of publication, country of origin, ethnicity on which the analysis was conducted, numbers of cases and controls, and frequency of genotypes in case and control groups. Ethnicities were divided into Asians, Caucasians, and Hispanics.P > 0.05 meant that the control group satisfied HWE. Pooled ORs combined with 95% CI were calculated to detect the potential correlation between this polymorphism and the IDD based on allelic effect (T vs. C), homozygote (TT vs. CC), heterozygote (TC vs. CC), dominant (TC/TT vs. CC), and recessive (TT vs. TC/CC) models, respectively. The Z test was performed to estimate the statistical significance of the pooled effect. The heterogeneity was evaluated by a Chi-square-based Q statistic test via I2 statistic and P value. No significant heterogeneity was detected if I2 < 50%, and the fixed effect model was used to calculate the pooled OR. Otherwise, the random effect model was performed. The origin of heterogeneity was detected through a subgroup analysis and a sensitivity analysis. The subgroup analysis was performed based on ethnicity, whereas the sensitivity analysis was performed by removing each article of significant heterogeneity. Publication bias was estimated using the Egger’s regression test. The statistical analysis was performed by STATA12.0 . All two-tailed P values were computed and statistical significance cut-off was 0.05.Chi-square analysis was employed to investigate the potential deviation of HWE and JZ and MY designed the study and extracted data, JZ and JS performed the literature search and formulated inclusion and exclusion criteria, JZ performed the statistical analysis. YB and JZ drafted the manuscript, ML revised this manuscript. All authors read and approved the final manuscript.The authors have declared no competing interests.


Background. The results from published studies regarding association of transcription factor 7-like 2 (TCF7L2) variant rs7903146 with dyslipidemia have been conflicting and inconclusive. Methods. We carried out a meta-analysis that aimed to investigate the association of the rs7903146 variant with plasma lipid levels using electronic database and published studies. Data was extracted by a standard algorithm. Dominant, recessive, homozygote, and heterozygote comparison models were utilized. Results. 24 studies incorporating 52,785 subjects were included in this meta-analysis. Overall, the minor allele (T) was associated with lower risk for hypertriglyceridemia in subjects with type 2 diabetes , P = 0.048, Pheterogeneity = 0.47; recessive model: SMD = −0.10, 95% CI , P = 0.01, Pheterogeneity = 0.56). No association was found between minor (T) allele and plasma TC, LDL-c, or HDL-c levels in subjects with type 2 diabetes or metabolic syndrome (MetS) and no association was found between minor (T) allele and plasma TG levels in nondiabetic subjects. Conclusions. Our meta-analysis indicated the association between TCF7L2 rs7903146 polymorphism and low plasma triglyceride (TG) level in subjects with type 2 diabetes. No association was found between rs7903146 variant and plasma lipids in nondiabetic subjects. Dyslipidemia is a major risk factor for coronary heart disease and stroke , 2. DespThe transcription factor 7-like 2 gene (TCF7L2), a Wnt signaling pathway effector, has been shown to be involved in the differentiation of adipocytes, regulation of adipokines, and pancreatic beta-cell function . PolymorWe systematically searched PubMed, Cochrane Library, Embase, reviews, and reference lists of relevant manuscripts before February 2014 by using Medical Subject Heading (MeSH) terms “Transcription Factor 7-like 2” or “TCF7L2” or “TCF7L” alone or paired with the following terms: “polymorphism,” “variant,” “SNP,” or “mutation.” The following four plasma lipids were included in our meta-analysis: TC, TG, LDL-c, and HDL-c.The selection criteria for eligible studies were the following: (1) studies had to include data reported on at least one of the four fasting plasma lipids; (2) mean plasma lipid levels and standard deviations (SD) or standard errors were available; (3) studies included genotyping data; (4) study subjects were 18 years of age or older; (5) baseline data before intervention were available for interventional studies. Animal studies, case reports, review articles, abstracts, reports with incomplete data, and family-based studies were excluded.The Newcastle-Ottawa Scale (NOS) was used to assess the quality of the studies. The NOS contains eight items categorized into three dimensions including selection, comparability, and exposure for case-control studies. In our meta-analysis, the NOS ranged from four to nine stars.Two investigators (Shuxia Wang and Kangxing Song) extracted data independently and discrepancies were resolved by consensus including a third author. Data were extracted using standardized methods. The following data were collected: article reference, first author, publication year, demographics including racial backgrounds, gender, age, health condition, and fasting status of the study subjects, mean and SD or standard error for the genotyping studies, genotyping methods, lipid assays, and unit of measurements.P values greater than 0.10 of the χ2-based heterogeneity Q-tests, and random effect model was used for P values less than 0.10. The I2 heterogeneity estimates were calculated and divided into 4 groups . The units of plasma lipids were converted from mmol/L to mg/dL, whenever necessary.Stata software  and Review Manager 5.0 software  were used for the meta-analysis. Four different genetic models, including dominant model (TT + TC versus CC), recessive model (TT versus TC + CC), homozygote (TT versus CC), and heterozygote comparisons (TC versus CC), were used in our analysis. All data in this analysis were presented as mean ± SD. The SD was derived whenever the standard errors were reported. A pooled standardized mean difference (SMD) together with 95% confidence interval (CI) was used for this meta-analysis. SMD was selected to eliminate the effect of multiple scanners used for plasma lipid measurements . A fixedP < 0.1 was used as an indication for significant publication bias.Funnel plots were used to evaluate publication bias. The funnel plots are asymmetric when there is publication bias. Egger's test, using MIX 1.7 software, was performed to estimate the degree of asymmetry of funnel plots. A stringent threshold of A total of 490 articles were identified searching PubMed, Embase, and Cochrane Library databases and from a manual approach (search of previous studies cited in the reviews and the reference lists of identified articles); 461 articles were excluded because they were not conducted in human or did not meet the goals of this meta-analysis. Full text assessment of the 29 potentially relevant articles resulted in 24 eligible studies that met the inclusion criteria for the meta-analysis . The mosA total of 24 studies , 31, 37 P = 0.07, SMD = −0.02, and 95% CI          . No sign = 0.16) . Under t, −0.01) . The het = 0.03) . Under t, −0.02) . The het= 0.002) . No diff4, 0.01) . The het = 0.02) .We then performed the subgroup analyses of the study population under four different genetic models.P = 0.048, SMD = −0.04, 95% CI , Pheterogeneity = 0.47, but not in metabolic syndrome (MetS) subjects: P = 0.49, SMD = −0.06, 95% CI , Pheterogeneity = 0.96, or the nondiabetic subjects: P = 0.99, SMD = 0.00, 95% CI , Pheterogeneity = 0.48, or the general population: P = 0.27, SMD = −0.02, 95% CI , Pheterogeneity = 0.02 , Pheterogeneity = 0.56 , Pheterogeneity = 0.66, or the general population: P = 0.15, SMD = −0.04, 95% CI , Pheterogeneity = 0.001 , Pheterogeneity = 0.19), MetS subjects , Pheterogeneity = 0.54), nondiabetic subjects , Pheterogeneity = 0.77), or general population , Pheterogeneity < 0.00001)  .P = 0.25, SMD = −0.05, 95% CI , Pheterogeneity = 0.01) or MetS subjects , Pheterogeneity = 0.76), nondiabetic subjects , Pheterogeneity = 0.94), and general population , Pheterogeneity = 0.02)  .http://dx.doi.org/10.1155/2015/468627).We performed the meta-analysis of the association of TCF7L2 rs7903146 polymorphism with TC, HDL-c, and LDL-c. There were no differences between rs7903146 polymorphism with TC, HDL-c, and LDL-c under 4 different genetic models  .Publication bias was assayed by visual funnel plot inspection and Egger's test. The funnel plots comparing the differences in TC, TG, LDL-c, and HDL-c were all symmetric  has a protective effect against plasma TG but not TC, LDL-c, and HDL-c levels. There was no association between the minor allele and plasma lipids in nondiabetic subjects.TCF7L2 rs7903146 is the most common susceptibility variant for type 2 diabetes across the world , 39. It These results are, however, consistent with earlier findings in animal models. These studies have suggested that TCF7L2 may regulate plasma lipids through insulin-independent pathways. We have shown in LRP6 mutant mice that impaired Wnt signaling is associated with reduced TCF7L2 expression, increased de novo lipogenesis, and cholesterol synthesis and secretion in the liver, leading to high plasma TG levels. Reduced Wnt/TCF7L2 activity in these mice results in IGF1-dependent but insulin-independent activation of AKT-mTOR-SREBP axis . Thus, iHeterogeneity was observed in some subgroups using certain models. In subgroup analysis for the association between risk allele and TG, heterogeneity was found in diabetic subjects under heterozygote model and in general subjects under all four different genetic models. For the analysis of association between LDL and the risk allele, the heterogeneity was only found under dominant model in subjects with type 2 diabetes and MetS subjects. The association analysis of HDL with risk allele showed heterogeneity for subjects with type 2 diabetes and general subjects under dominant, recessive, and heterozygote models. There was no heterogeneity in subgroup analysis of TC with risk allele. Random effect models were used to decrease the effect of heterogeneity in subgroup analysis. The present analysis had several limitations. First, most of the included studies were designed to study the association of TCF7L2 variants with diabetes , 29, 31,In conclusion, our meta-analysis identified an association between T genotype of TCF7L2 rs7903146 with lower TG level in subjects with diabetes. This finding identifies TCF7L2 as a potential target for development of novel therapeutics against hyperlipidemia.Supplement Figures 1-3 Forest plots of the TGCF7L2 rs7903146 polymorphism and total cholesterol, LDL-cholesterol, and HDL-cholesterol association. Under different models: A: dominant.Model, TT+TC vs. CC; B: recessive model, TT vs. TC+CC; C: Homozygous, TT vs. CC; D: Heterozygous, TC vs. CC.Supplement Figure 4: Funnel plot of TGCF7L2 rs7903146 polymorphism and total cholesterol, LDL-cholesterol and association under different models: dominant model (A) recessive model (B), homozygous recessive model (C) and heterozygous model (D).


MTHFR affect the activity of the enzyme. Genetic associations have been related to treatment efficacy. Abnormal levels of the enzyme methylenetetrahydrofolate reductase (MTHFR) are associated with an increased risk of both cardiovascular and cerebrovascular disease and higher concentrations of homocysteine. Abnormal levels are also related to birth defects, pregnancy complications, cancer and toxicity to methotrexate (MTX). Polymorphisms of  To establish the frequency of the C> T polymorphism at nucleotide 677 of the MTHFR gene in a group of Colombian individuals.®). The frequency of the C> T MTHFR rs1801133 marker polymorphism was analyzed. Data from pharmacogenetic microarrays that include MTX sensibility-associated polymorphisms were retrospectively collected , as were C/T and T / T (p= 0.0001). Microarray data from 68 men and 84 women were analyzed. Comparisons of genotype C/C vs. C/T and T/T were statistically significantly different (MTHFR polymorphisms and might present toxicity and adverse effects if treated with MTX, which suggests the need to evaluate therapeutic alternatives based on individual pharmacogenetic studies. Results for the C/C and C/T genotypes in a Colombian population are similar to other previously studied groups of healthy subjects. Subjects from our population might be at risk of developing diseases associated with  The sequencing of the human genome and the study of single nucleotide polymorphisms (SNPs) have generated a considerable amount of data that reveal the basis of the genetic association of hundreds of diseases and toxicity, as well as the efficacy of various drugs. The latter area of study is called "pharmacogenetics". The technology applied to reveal these associations is available, and costs are becoming progressively reduced in relation to the benefit of optimization and clinical application of their findings.  The response of each individual to the same treatment can be in part, to heritable factors, creating differences in drug efficacy among subjects due to polymorphisms in genes that encode enzymes involved in the metabolism of certain drugs, in their transporters and/or specific treatment targets  A gene is described as "polymorphic" when allelic variants are present. The activity of the protein can vary when compared to the original allele. Pharmacogenetics may additionally explain individual differences in response to treatment and may have predictive value for patient response to various drugs MTHFR gene related to the efficacy of MTX have been studied. The C677T polymorphism enzyme activity was reduced by 35% in carriers and has a prevalence of 40% in the population. MTX activity is reduced 50-70% in homozygous TT at position 677 with a prevalence of 8-10% in the population  A classic example of pharmacogenetics is rheumatoid arthritis (RA), an autoimmune disease that affects between 0.5% and 1% of the population. One of the most widely used therapies in this disease, which is one of the most effective from the clinical standpoint, is methotrexate (MTX). Several reports describe its use since 1951, and clinical trials show its effectiveness but also demonstrate its toxicity, including increased nodes, pneumonitis, neurologic involvement, gastrointestinal complications such as nausea, vomiting and diarrhea, increased transaminases, hematologic abnormalities, skin rash, stomatitis and alopecia  Additionally, MTX inhibits dihydrofolate reductase (DHFR) and folate-dependent enzymes such as timidilatosintase (TS) and methylenetetrahydrofolate reductase (MTHFR). It has been shown that their polymorphisms affect enzyme activity, resulting in elevated homocysteine levels and toxicity et al.  The study of the MTHFR enzyme has acquired much interest after the fundamental discovery by Kang MTHFR gene alone, or in the context of molecular genetic profiles as part of preventive medicine. These profiles evaluate pharmacogenetic associations with drugs, including MTX. The polymorphism analysis can also be performed by direct sequencing of the entire gene or a fragment there of, by analysis using restriction enzyme polymorphisms (RFLP) or by microarray techniques. The spectrum of the frequency of the MTHFR gene polymorphism C677T in a group of healthy Colombian individuals was determined to evaluate the genetic background in relation to disease susceptibility and pharmacogenetic applications.  Genetic analysis can be requested for the  A retrospective cross-sectional study in 152 healthy Colombian individuals over 18 yrs was conducted using molecular genetic profiling. DNA samples were collected from saliva samples in the area of preventive medicine of the "Instituto de Referencia Andino", including all applications from medical orders and/or direct individuals received between the years 2012 and 2014. All individuals signed an informed consent. Subjects for whom demographic data were not complete were excluded. ® in Medication DNA InsightTMgenetic test evaluates 15 metabolism associations for drug metabolism, including MTX in addition to Abacavir® hypersensitivity, aminoglycoside-induced ototoxicity, response to beta blockers, carbamazepine hypersensitivity, clopidogrel metabolism, estrogen supplementation, alpha interferon, metoprolol metabolism, metabolism and hypersensitivity to phenytoin, proton-pump inhibitor, simvastatin induced myopathy, metabolism of voriconazole and warfarin.  The pharmacogenetic polymorphism panel from PathwayGenomicsMTHFR gene. Each microarray position contains 10-12 picomoles of a specific DNA sequence under high stringency. Probe-target hybridization was detected and quantified by fluorophores attached to the probe. The frequency of the C677T polymorphism, consisting of a C>T substitution in the MTHFR gene marker rs1801133, was analyzed. DNA microarrays were used to measure expression levels of probes, corresponding to allele mutations "C" and "T" in the ® statistical program for Windows® V20. They were not stratified by ethnic group or socioeconomic levels.  Each participant was identified by a code used in a database, where the descriptive analysis for continuous variables such as age was performed with measures of central tendency and Shapiro Wilk normality test. The description of categorical variables  was performed by frequency analysis. Given the variable nature of the study association levels, they were evaluated by a Chi square test (X²) with a confidence level of 95%. In addition, Hardy Weinberg equilibrium was tested. The results were transferred to the SPSSStudy was approved by the Ethics Committee of Fundacion Instituto de Reumatología Fernando Chalem. The study included 152 individuals, 68 men (44.7%) and 84 women (55.3%) aged 40 ±13 and 41 ±13 yrs, respectively. The origins of the samples were as follows: Medellin , Cali , Bogota , Barranquilla and Cartagena (6 subjects: 3.9%), Pasto .p = 0.001 and p= 0.026, respectively), as were C/T and T/T p= 0.0001 , followed by the homozygous C/C with 52 individuals (34.2%). The homozygous genotype T/T had the lowest frequency in the population studied , corresponding to 13.2%. The frequencies of genotype C/C vs. C/T and T/T were significantly different  (p= 0.211).  No statistically significant associations were observed between genotype and allele with sex, est (X²) . The pop In assessing the allele frequency in the population, the C allele was found in 86.3% (n= 306) of individuals and the T allele in 13.7% (n= 42). MTHFR gene polymorphisms are among the most studied  There are functional polymorphisms in genes encoding enzymes that are involved in metabolism that may produce susceptibility to diseases. For that reason, MTHFR gene, and therefore, there is no evidence of selection for any of them. Because it has been found that this polymorphism affects MTHFR enzyme activity, it is important to assess its frequency and its effect on the population. In the current study, the population was in Hardy-Weinberg equilibrium for the different alleles of the MTHFR gene have been described, but two main variants from the functional point of view are the most studied: A1298C and C677T. The organic effect of these polymorphisms has been determined, generating predictive results for patient response to MTX The MTHFR enzyme is a critical protein in the metabolism of 5-methyl tetrahydrofolate reductase, which is the donor in several biochemical reactions involved in the methylation of homocysteine. A dozen polymorphisms of the Most genotype frequency observed in the population corresponded to the heterozygous C/T, followed by C/C, with the lowest frequency for the T/T genotype. The C677T polymorphism produces a change to alanine from valine at codon 222, corresponding to a thermolabile MTHFR enzyme variant that generates a decrease in activity and an increased plasma level of homocysteine 20. The homozygous genotype T/T has 30% of the original activity with a prevalence of 8 to 10% in the general population. Heterozygotes have approximately 60% of normal activity and a prevalence of 40% in the population MTHFR gene polymorphisms in women with and without cervical cancer was evaluated, showed a C/C genotype in 22.4% of the 89 control women with an average age of 44 years old, lower than the 32.1% found in the present study. Moreover, genotype C/T was present in 55.1% of the study population, similar to that found in the group of Colombian women analyzed (53.6%). Finally, the T/T genotype was found in 22.5% of the population, a figure significantly higher than in the present study, considering that this is the homozygous variant that is associated with an increased risk of cancer. Allele frequencies of both the C and T alleles were 0.5 in the Mexican study A study conducted in Mexico, in which the frequency of Several previous studies have been conducted in Colombia et al. studied 102 volunteers of both sexes (53 women and 49 men) from various parts of Colombia residing in Bogota, aged between 18 and 50 yrs old Bermudez In a study of a population sample of 206 students in Bogota, Colombia, overall frequencies were calculated using data from healthy controls reported in other studies, finding similar frequencies of genotypes and alleles detected in the group of subjects analyzed in our study The frequency of TT homozygosity in other populations worldwide varies from 0% to over 30%  In summary, substantial variability has been observed in the frequencies of C and T alleles worldwide. In Colombia, some studies show a predominance of allele C and others of allele T. Differences between populations suggest that both allele frequencies in certain circumstances could produce selective advantages and disadvantages. Some reports suggest that the T allele can produce resistance to infections such as malaria and cytomegalovirus and may also protect against hypertension  The results of the present study are limited to a Colombian population who applied to study their genetic profile in the cities of Bogota, Medellin, Barranquilla and Cali, which does not afford a comprehensive understanding of the Colombian population. The findings in different studies have shown variation, even when people have been analyzed in the same geographical area. It is possible that the differences found in the different studies may have resulted from different methodological approaches used and the ethnic heterogeneity of the subjects MTHFR gene polymorphism genotypes associated with toxicity and adverse defects to MTX could be at high risk, suggesting the need to evaluate therapeutic alternatives with pharmacogenetic studies of MTX genotype T/T. From the clinical perspective, the classification of patients for polymorphisms of the MTHFR gene before administering medication would offer alternative treatments without subjecting patients to harmful effects, optimizing therapy and reducing costs for health systems.  Some subjects in our population with  The results for the genotypes C/C and C/T in Colombia are as variable as in other groups of healthy individuals studied in other populations, and this variability may be subject to selective pressures of the environment in different regions of Colombia.


CYP2C19 gene polymorphisms and various environmental factors and their interactions on the risk of esophageal squamous cell carcinoma (ESCC) in a Chinese Han population.The purpose of this study was to explore the effects of A 1:2 frequency-matched case control study of 285 patients and 570 controls was conducted from June 2010 to May 2011 in AnXi of Fujian province, China. Environmental factors were investigated using a self-administered questionnaire and genotypes were determined using polymerase chain reaction restriction fragment length polymorphism based methods. Unconditional logistic regression models were used for statistical evaluation.CYP2C19*2 GA/AA genotype was significantly more prevalent in ESCC patients and individuals with at least one copy of the CYP2C19*2 A allele had a 3.19-fold increased risk : 2.21–4.61, P < 0.001) of ESCC compared with those without this allele. We found no significant associations between CYP2C19*3 genotypes and ESCC. The Cyp2C19*2 polymorphism appeared to have a multiplicative joint effect with tea drinking and hot beverage/food consumption  and an additive joint effect with pickled vegetable consumption .Current or former smoking, consumption of pickled vegetables or hot beverages/food, having a first degree relative with ESCC and history of reflux esophagitis were significantly associated with increased ESCC risk, whereas tea drinking and consumption of fresh vegetables and fruits were significantly associated with decreased risk. The CYP2C19*2 polymorphism plays an important role in the development of ESCC in the Chinese population, modified by tea drinking and consumption of pickled vegetables or hot beverages/food. Further studies are warranted to confirm our results.Our findings suggest that the The online version of this article (doi:10.1186/s12881-015-0156-3) contains supplementary material, which is available to authorized users. Esophageal squamous cell carcinoma (ESCC), a treatment resistant cancer that can withstand a combination of surgery, chemotherapy and radiotherapy , is the CYP2C19 gene polymorphisms may play a major role in inter-individual variability in drug response, drug–drug and drug–xenobiotic interactions and susceptibility to chemical-induced diseases.Cytochrome P450 2C19 (CYP2C19) is an important phase I enzyme expressed abundantly in endothelial and smooth muscle cells . This enCYP2C19 are known to be associated with reduced enzyme activity; however, most cases are due to either CYP2C19*2, which is characterized by a point mutation in exon 5 , or CYP2C19*3, which has a mutation in exon 4  [CYP2C19*2 or CYP2C19*3 are considered poor metabolizers (PMs), whereas those with at least one CYP2C19*1 allele are classified as extensive metabolizers (EMs) [CYP2C19 functional polymorphisms as modulators of susceptibility to cancer, inconsistent findings have appeared in the literature; for example, CYP2C19 PMs have a high incidence of stomach cancer and lung cancer [CYP2C19*3 than Caucasians [CYP2C19*2 and CYP2C19*3 have been shown to account for almost 100% of Asian PM alleles. Therefore, it would be interesting to determine whether the high incidence of ESCC is correlated with a greater frequency of CYP2C19*3 or CYP2C19*2 in the Chinese population.Several polymorphisms of 4986893) . The prers (EMs) . Despiteg cancer , but cong cancer . Furtherucasians ,16 and tCYP2C19*2 or CYP2C19*3 polymorphism may have a higher risk of ESCC. We also speculated that there should be a synergistic interaction between the effect of environmental factors and that of the genetic variation in the risk of ESCC.In the present study, we hypothesized that individuals with the This study was approved by the institutional ethical committees of Fujian Medical University. Written informed consent was obtained from all participants before their participation in the study. All investigations performed in this study were conducted in accordance with the guidelines of the 1975 Declaration of Helsinki.A 1:2 frequency-matched case control study was performed between June 2010 and May 2011 in AnXi, in the Fujian Province of China. Two hundred and eighty-five patients  were diagnosed with histologically confirmed ESCC at AnXi Hospital. Five hundred and seventy cancer-free control subjects  were chosen randomly during the same period from among local residents who underwent a routine health check and were free from any known major diseases. Controls were frequency matched with ESCC patients according to sex, age (within 5 years), ethnicity and area of residence. All subjects were genetically unrelated ethnic Han Chinese from AnXi or the surrounding regions.A standard questionnaire was administered to cases and controls by specially trained interviewers. Questions covered demographic characteristics , dietary habits, lifestyle habits such as tobacco smoking and alcohol drinking, personal medical history and family history of cancer. Non-smokers were defined as individuals who had never smoked cigarettes or who had smoked less than 100 cigarettes in lifetime. Ever tea drinking was defined as drinking at least 1 cup of green tea per week for more than 6 months. Ever alcohol drinkers were subjects who had consumed any alcoholic beverage, including beer, wine or distilled spirits, at least once per week for a minimum of 6 months.CYP2C19*2  and CYP2C19*3  genotypes were determined using polymerase chain reaction (PCR)-based restriction fragment length polymorphism. PCR was performed with 25 μl of reaction mixture containing 100 ng of DNA, 0.1 mmol/l of each primer, 0.2 mmol/l of deoxynucleoside triphosphate, 1.0 U of Taq DNA polymerase (Takara Bio Inc.), 1 × reaction buffer and 1.5 mmol/l of MgCl2. The PCR profile comprised an initial melting step for 2 min at 95°C, followed by 35 cycles of 30 s at 94°C, 30 s at 58°C and 30 s at 72°C and a final elongation step of 10 min at 72°C. The forward primer 5′-AATTACAACCAGAGCTTGGC-3′ and the reverse primer 5′-TATCACTTTCCATAAAAGCAAG-3′ were used to detect the CYP2C19*2 allele and the PCR products digested with SmaI I ; the 168 bp PCR products were cut into 117 and 51 bp fragments in the wild type (WT) but not in CYP2C19*2. The forward primer 5′-AAATTGTTTCCAATCATTTAGCT-3′ and the reverse primer 5′-ACTTCAGGGCTTGGTCAATA-3′ were used to detect the CYP2C19*3 allele and the PCR products digested with BamHI (New England Biolabs); the 271 bp PCR products were cut into 175 and 96 bp fragments in the WT but not in CYP2C19*3. The digested PCR products were analyzed on 3% agarose gels and stained with ethidium bromide. Individuals who inherit two mutant CYP2C19 alleles, whether of the same type  or one of each (*2/*3) have a reduced capacity to metabolize CYP2C19 substrates and are considered to be PMs. Individuals who are homozygous (*1/*1) or heterozygous  for the WT CYP2C19*1 allele have an effective enzyme for metabolizing CYP2C19 substrates and are EMs. For quality control, genotyping was performed by laboratory personnel blinded to the case control status and blank controls were included in each plate. In addition, 10% of the samples were randomly selected and genotyped a second time; the concordance was 100%.Blood samples were collected with a standard venepuncture technique and ethylenediaminetetraacetic acid-containing tubes. DNA was extracted from the blood cell pellet using a Blood Genome DNA Extraction Kit  and stored at −20°C. t-test for continuous variables. Adjusted odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated to evaluate the associations of lifestyle habits including smoking, alcohol/tea drinking and diet with risk of ESCC. All models were adjusted for age as a continuous variable, sex, education, income, occupation (farmer/other), family history of cancer in first degree relatives and other potential confounding factors. P values for OR trends were calculated using the order of each risk factor category as a categorical variable.Differences in the distribution of demographic characteristics and other risk factors for ESCC between patients and controls were tested using the chi-square test for category variables and Student’s CYP2C19 polymorphisms was in Hardy–Weinberg equilibrium among the control subjects. Associations between CYP2C19 genotypes and ESCC risk were evaluated using unconditional logistic regression with adjustment for potential confounding factors including age, sex, education, income, occupation, family history of cancer, consumption of vegetables, fruits and meat, smoking status (no/yes), alcohol drinking (no/yes) and tea drinking (no/yes).A goodness of fit chi-square test was used to assess whether the genotype distribution of P values for multiplicative interactions were derived from a cross-product term for gene and environmental exposure introduced into a multiplicative model. Interaction contrast ratio (ICR) was used to evaluate potential additive interactions, as follows: ICR = OReg – ORe – ORg + 1, where OReg is the OR for both the genotype and the environmental exposure, ORe is the OR for the environment only and ORg is the OR for the genotype only. For ICR > 0, we concluded that there was a positive additive interaction. Ninety-five percent CIs for ICRs were calculated according to the method of Hosmer [Stratified analysis was used to explore potential gene–environment interactions. We dichotomized the genetic polymorphisms by grouping subjects into carriers and non-carriers of the risk genotype. Similarly, environmental factors were dichotomized by appropriate grouping. f Hosmer ; an ICR P values of < 0.05 were considered statistically significant.All statistical analyses were performed using R software . Two-sided P > 0.05), indicating that the frequency matching was adequate. However, a greater proportion of farmers and a lower education level were observed among the patients compared with the controls (both P < 0.001).Associations between demographic characteristics and ESCC are shown in Table Table CYP2C19*2 A and CYP2C19*3 A alleles were 0.335 and 0.054, respectively, and were in Hardy–Weinberg equilibrium (both P > 0.05). On single allelic analysis, tests for association between ESSC and the two SNPs showed a significant difference only for CYP2C19*2 (G681A); the association remained significant after multiple comparison correction by permutation tests , with GA and AA being more frequent in patients than in controls. The homozygous mutant genotype (AA) of CYP2C19*3 was not detected in any subject, patient or control, and the frequencies of the CYP2C19*3 GG and GA genotypes did not differ significantly between the controls and patients.Single nucleotide polymorphism (SNP) data, observed allele frequencies and Hardy–Weinberg test results are presented in Table CYP2C19 polymorphisms and risk of ESCC. After adjustment for age, sex, income, marital status, education, smoking, tea drinking and other variables, a significant risk effect for ESCC was found to be associated with the CYP2C19*2 genotype. Specifically, compared with homozygous GG subjects, carriers of the heterozygous GA and homozygous AA genotypes had a significantly increased risk of ESCC  in an allelic dose–response manner (adjusted PTrend = 0.000). On pooled analysis, we found that individuals with at least one copy of the CYP2C19*2 A allele had a 3.19-fold increased risk  of ESCC compared with those without this allele. We found no significant associations between the CYP2C19*3 genotype and ESCC in multivariate logistic regression models.Unconditional logistic regression analysis was used to evaluate associations between the genotypes of the two *2/*2 and *2/*3 but not *3/*3 were found in this study) was significantly higher among the ESCC patients than in the control group (20.70% vs 13.86%). After adjustment for age, sex, income, marital status, education, smoking, tea drinking and other variables, CYP2C19 PMs had a 1.71-fold increased risk of ESCC compared with EMs.The association between being a CYP2C19 PM and having ESCC was further analyzed. The frequency of CYP2C19 PMs  and an additive joint effect with pickled vegetable consumption . Specifically, tea drinking may decrease the effect of the GA/AA genotype on ESCC risk. By contrast, consumption of hot beverages/food or pickled vegetables may increase the risk effect of the GA/AA genotype.We also explored the combined effects of the CC Table . No signCYP2C19 gene (CYP2C19*2 and CYP2C19*3) in the development of ESCC in a Chinese population, including the effects of environmental risk factors. We found CYP2C19*2 to be the more common locus in the Chinese population, and multivariable logistic analysis revealed that the presence of the CYP2C19*2 A allele (AA or AG genotype) increased the risk of ESCC. Most of the established risk factors for ESCC evaluated in the present study had strong associations with ESCC risk. Generally in case control studies, a potential gene–environment interaction is assessed; our results show that the association between the CYP2C19*2 A variant and ESCC was modified by tea drinking and consumption of pickled vegetables or hot beverages/food. These findings suggest that CYP2C19 is involved in the detoxification of certain carcinogens involved in the development of ESCC. To our knowledge, this is the first study to report this gene–environment interaction between environmental factors and the CYP2C19*2 polymorphism with respect to the risk of ESCC in a Chinese population.ESCC is prevalent among the Chinese population, with marked regional variations in incidence and mortality. Although the pathogenesis of ESCC is not fully elucidated, accumulative epidemiologic evidence has shown that genetic and environmental factors play crucial roles in its etiology. In the present study, we conducted a case control study to examine the role of the two most common functional variants of the CYP2C19 gene is highly polymorphic; the most important alleles are CYP2C19*2  and CYP2C19*3 . The nucleotide changes in CYP2C19*2 and *3 lead to a splicing defect and a stop codon, respectively, and thereby to nonfunctional proteins and the PM phenotype [CYP2C19 are suspected to be associated with susceptibility to cancer, though several studies concerning CYP2C19 polymorphism and cancer susceptibility among various populations have reported inconsistent results [CYP2C19*2 to be higher than that of CYP2C19*3 in Chinese Han subjects and the PM genotype was associated with an increased risk of ESCC. This finding is consistent with a recent meta-analysis by Wang et al. [CYP2C19 PM genotype and increased risk of esophageal cancer  among 308 cases and 644 controls. However, our single-locus analysis found a significantly elevated risk of ESCC for the CYP2C19*2 allele but not for CYP2C19*3. Subjects carrying the CYP2C19*2 A allele (AA or GA genotype) had a higher risk of ESCC than GG and patients carrying at least one variant allele had a 3.19-fold increased risk of developing ESCC. CYP2C19*3 has a premature stop codon in exon 4 and it is biologically plausible that the CYP2C19*3 polymorphism is a modulator of cancer susceptibility; however, we did not find CYP2C19*3 to be associated with ESCC susceptibility in our study population. This finding is inconsistent with a recent report from Shi et al. [CYP2C19*3 A allele was significantly higher in ESCC patients than in controls . Our current understanding of carcinogenesis indicates a multifactorial and multistep process involving various genetic alterations and environmental factors and it is unlikely that risk factors for cancer act in isolation from each other. Therefore, differences in certain environmental factors may have contributed to this discrepancy. For example, Shi et al. [CYP2C19, one of the most important cytochrome P450s, is a key enzyme that is not only responsible for the metabolism of numerous therapeutic drugs -20 but ihenotype . The two results -26. In tg et al. , who foui et al. , who foui et al.  found thCYP2C19 polymorphism is considered to be one of the factors that determine an individual’s susceptibility cancer through variations in ability to detoxify carcinogens and/or activate procarcinogens [CYP2C192 gene and environmental factors on ESCC risk. Our findings suggest that the CYP2C19*2 polymorphism has a multiplicative joint effect with tea drinking and hot beverage/food consumption and an additive joint effect with pickled vegetable consumption. Specifically, consumption of hot beverages/food or pickled vegetables may increase the risk effect of the GA/AA genotype in ESCC. By contrast, tea drinking may decrease the risk effect of the GA/AA genotype.cinogens ,29. CYP2cinogens . To explCYP2C19*2. In individuals carrying the CYP2C19*2 GA/AA genotype, the risk of ESCC in those who consumed picked vegetables was almost twofold greater than that of those who did not. The mechanisms by which pickled vegetables and the CYP2C19*2 polymorphism interact to influence the development of ESCC are unknown; however, previous studies have shown that the traditional method of preparing pickles by packing moist vegetables in jars for weeks or months allows fermentation and growth of fungi and yeasts [CYP2C19*2 polymorphism and pickled vegetable consumption may be expected.Ecologic studies have shown higher risks of esophageal and gastric cancers in areas where the consumption of pickled food is high ,32. Consd yeasts ,34 and cd yeasts -37. Furtd yeasts . TherefoCYP2C19*2 polymorphism. The mechanisms by which drinking beverages at high temperatures and the CYP2C19*2 polymorphism interact to influence the development of ESCC are unknown and additional studies are warranted to explain and confirm this preliminary evidence.Numerous experimental and clinical studies have suggested that drinking beverages at high temperatures is a cause of esophageal cancer ,40. MoreCYP2C19*2 polymorphism. Among individuals with the CYP2C19*2 GA/AA genotype, the risk of ESCC in tea non-drinkers was almost twice that in tea drinkers. Polyphenols in tea possess potent antioxidant and anti-inflammatory properties and modulate several signaling pathways, and these biochemical features are responsible for tea’s anticancer properties [CYP2C19*2 GA/AA genotype. Intervention trials are needed to provide more convincing evidence.The results of the present study also suggest possible interactions between tea drinking and the operties ,43. Prevoperties . CYP2C19CYP2C19 polymorphism is a single example that was previously reported to have potential functional significance. Further studies of additional SNPs of functional significance are warranted to identify the role of CYP2C19 polymorphism and gene–environment interactions in esophageal carcinogenesis.The potential limitations of the present study should be considered. First, this was a hospital-based case control study and selection bias may exist because the control subjects were recruited from a healthy population undergoing an examination, which may not accurately represent a geographically matched population with exposure to similar environmental factors. However, the control subjects came from the same region as the patients and were sampled randomly, which may have reduced the selection bias. Second, recall bias is inevitable in case control studies. However, this would not affect the genotype data and is therefore of less concern in the study of gene–disease associations. Finally, the selected CYP2C19*2 A allele (AA or AG genotype) plays an important role in the development of ESCC in the Chinese population. In addition, our results show that the association between the CYP2C19*2 A variant and ESCC was modified by tea drinking and consumption of pickled vegetables or hot beverages/food. Since our study has limited sample size, further studies in a large population are needed to confirm these findings.In summary, our findings suggest that the


Usually the genetics of human longevity is restricted to the nuclear genome (nDNA). However it is well known that the nDNA interacts with a physically and functionally separated genome, the mitochondrial DNA (mtDNA) that, even if limited in length and number of genes encoded, plays a major role in the ageing process. The complex interplay between nDNA/mtDNA and the environment is most likely involved in phenomena such as ageing and longevity. To this scenario we have to add another level of complexity represented by the microbiota, that is, the whole set of bacteria present in the different part of our body with their whole set of genes. In particular, several studies investigated the role of gut microbiota (GM) modifications in ageing and longevity and an age-related GM signature was found. In this view, human being must be considered as “metaorganism” and a more holistic approach is necessary to grasp the complex dynamics of the interaction between the environment and nDNA-mtDNA-GM of the host during ageing. In this review, the relationship between the three genetics and human longevity is addressed to point out that a comprehensive view will allow the researchers to properly address the complex interactions that occur during human lifespan. Longevity is a complex trait whose genetics has been extensively studied since many years. Understanding the genetic makeup that predisposes to longevity is an urgent challenge owing to the explosion of the elder population in western as well as in emerging countries.Usually the studies on the genetics of human longevity are restricted to the analysis of nuclear genome (nDNA). However, another essential genome, that is, the mitochondrial genome (mtDNA), is part of the genetic machinery of each cell. Despite its limited length, the mtDNA encodes for few genes that constitute a quantitatively relevant group because of the high copy number of mtDNA in each cell.These two genomes do not work in the void and life/survival, as well as ageing and longevity, depends on their complex interaction with environment/lifestyle. To this scenario we have to add another level of genetic complexity represented by the microbiota, that is, the whole set of bacteria that live in different anatomical districts of our body with their whole set of genes (microbiome). Indeed, the most comprehensive view is to consider human being as a “metaorganism” resulting from the close relationship with symbiont microbial ecosystems. A particular attention has been recently devoted to the gut microbiome (GM). The GM probably represents the most adaptable genetic counterpart of the human metaorganisms, being extremely plastic in response to age-related physiological changes in diet and modification in lifestyle.Thus, the result of the ageing process is defined by the sum of a number of factors both biological and nonbiological . Therefore while the ageing research based on the study of animal models starts assuming the existence of major genes that determine longevity, in humans this assumption represents an oversimplification. The study of human model imposes a more holistic view of the genetics to grasp the complex dynamics of the interaction between the environment, stochasticity, and the three genetics of the host .The main aim of this review is to sum up the state-of-the-art of the knowledge of the three genetic components in human longevity to demonstrate that within this comprehensive view the genetics moves from a crystallized concept (genes are forever) to a much more flexible and dynamic perspective, in which the complex interaction between genetic makeup and environment across the long-lasting human lifespan is properly addressed.The study of genetics of ageing in human being is tangled given the high complexity of the interaction between heterogeneous environmental factors and genetic variability across a long period of time. A strategy for disentangling this complexity is to focus on robust human models of longevity such as centenarians.Centenarians are a model of successful ageing as in most cases they display medical histories free from most of the major age-related diseases, including cancer, dementia, diabetes, and cardiovascular diseases. Their ability to reach the extreme limit of human life—escaping, or largely postponing, age-associated pathologies—is the result of the combination of a well-preserved and efficient immune system, a good capability to cope with different stressors, an appropriate lifestyle, and a robust genetic background –5. IL6 -174 C/G [ IL10 -1082 A/G [ PON1 gene [ SOD2 401nt T/C [ TP53 gene at codon 72 [At the beginning, studies on longevity were conducted on lower organisms and animal models, providing evidences that longevity could be influenced by many conserved genetic variants with small effects . Complyi1082 A/G , PON1 ge01nt T/C , the argcodon 72 –15, and codon 72 –21, but codon 72 .This can be due to different reasons that include the effect of population structure  and the Technological advances in the last 10 years have fostered the study of the genetics of complex traits by means of genome-wide approaches that allow the simultaneous analysis of thousands of genetic variants on large cohorts.Many genome-wide association studies (GWASs) have been conducted assuming that long-lived individuals could share several common genetic variants that influence human lifespan. APOC1 and is in linkage with APOE [Nebel et al. genotyped 1848 Germans, 763 individuals aged 94–110 years and 1085 controls aged 45–77 years, and replicated the results on an independent cohort of 1614 subjects. They reported a statistical significant association only for one SNP, rs4420638, that is located nearith APOE .P = 3.39∗10−17). This SNP is located in TOMM40 gene, close to APOE gene and, even if it displays only a moderate linkage withε4 APOE  determining variant, authors report an APOE dependent effect of rs2075650 on longevity.Deelen et al.  compared TOMM40 gene but the contribution of this variant to the predictive power of the genetic risk model is poor. This is probably because of the low frequency of GG genotype that is almost absent in centenarians (frequency: 0.1%) and that makes the prediction of lifespan of AG and AA carriers uncertain without further genetic information [Another GWAS was performed by Sebastiani et al.  considerormation . It is rSebastiani and colleagues tested the 281 identified SNPs on 5 studies of centenarians from USA, Europe, and Japan and they found that 128 SNPs reached statistical significance, bringing out biological pathways deeply involved in exceptional longevity .APOE is the only gene accounted as a “longevity determinant” by several independent GWASs.ε APOE variants have been extensively analyzed and the frequency of ε4 allele has been found decreased in long-lived subjects [ε4 APOE variant in the Danish 1905 Birth Cohort and they found that relative risk of ε4 allele does not increase linearly with age, supporting the idea that this allele exerts its deleterious effect also in the last decades of life [subjects –33 but tsubjects , 35. Rec of life .Overall, GWASs have proved to be rather disappointing for identifying genetic determinants of complex traits, that is, longevity or many age-associated diseases, and established loci account only for a small proportion of trait heritability. The lack of robust results can be attributed to different reasons that include, among others, the need to conduct these studies on hardly available large cohorts, the phenotypic heterogeneity of longevity, the gene-environment interactions, and the failure to identify both low frequency variants with large effects and rare variants. One point to take into account is that each cohort in GWASs could be characterized by population-specific genetic makeup. This phenomenon has been suggested by De Benedictis and Franceschi  from the TCF7L2 gene showed a progressive increase in the frequency of risk genotype (TT) from centenarians to diabetic patients who developed one or more complications and the strongest genotypic association was detected when diabetic patients were compared to centenarians (P = 9.066∗10−7). The use of centenarians in this kind of studies has proved to be useful to evaluate the biological relevance of genetic variants emerging from GWASs. Authors speculate that if a SNP is considered a statistically significant but “weak” risk factor for the disease and it is present at similar frequency in centenarians and in patients, its biological relevance can be assumed as negligible. On the contrary, if the frequency of a genetic risk variant is much higher in patients than in centenarians, it is likely that this SNP plays a consistent biological role in the pathogenesis of the disease under study [Studies carried out so far were unable to discriminate between two basic assumptions, that is, whether the ability to reach the extreme limits of human life is due to the presence of polymorphisms in “longevity genes” that promotes the achievement of old age or to the absence of harmful variants . The stuer study . It is aIn addition to centenarians, a widely used model to investigate longevity consists of long-lived families. Indeed, it was observed that siblings of centenarians have an increased relative risk of reaching 100 years, sustaining the existence of a heritable component of human longevity –44. This TOMM40, APOE, and APOC1 genes. The analysis ofε APOE variants in GEHA nonagenarians siblings showed thatε4 allele frequency was significantly lower than that reported for the geographically matched younger controls (6.8% versus 12.7%). In agreement with this finding, ε4 allele carriers have about 50% lower chance to become nonagenarians than the non-ε4 carriers .A genome-wide linkage analysis on 2118 European nonagenarian full sibships of the GEHA project was performed to identify chromosomal regions involved in longevity . By usin per se a cause of ageing, but rather a consequence of the age-dependent accumulation of mtDNA damage. Indeed, it is well known that mtDNA mutations increase with age, and recent findings show that this increment is likely due to errors in replication machinery or to unrepaired damage, placing ROS mutagenic effect in the background. Two studies support this hypothesis: (i) a study by Kennedy et al. [γ) lead to a high accumulation of mtDNA mutations and to a consequent premature ageing of mice that carry these defects.Several theories on ageing process and longevity posed mitochondria in a central position. Mitochondria produce the cellular energy through oxidative phosphorylation (OXPHOS) and many metabolic pathways are located in these organelles as well as the pathway controlling apoptosis. The two main mechanisms that link mitochondria to ageing are the mutagenesis of mitochondrial DNA (mtDNA) and the production of reactive oxygen species (ROS). The relative contribution of these two mechanisms and their interplay in the ageing process are still matter of debate, but a detailed analysis of the history of these theories is out of the scope of this review and it is well described elsewhere . Howevery et al.  showed ty et al.  showed tγ, PGC-1α,PPAR and UCPs) providing further support to the hypothesis that mitochondria play a central role in the ageing process [Recent GWASs on age-related diseases –59 have  process –62. In h process , 63–73.J is characterized by mutations in complex I genes, this result might explain previous contrasting findings emerging from association studies on J haplogroup and longevity [Recently, the association between recurrent or sporadic mtDNA mutations and longevity has been highlighted by the results of the GEHA project. In this project the control region of 3000 samples and the complete mtDNA of 1292 samples were sequenced including 650 ultranonagenarians (90+) and a comparable number of controls, enrolled in Denmark, Finland, Southern Italy, and Greece . A haploongevity , 76–78. ongevity , 79, 80.ongevity . MoreoveThe complex relationship between mitochondrial genetics and longevity has been further puzzled when somatic mtDNA variability is considered. Indeed in one cell many mitochondrial genomes exist and the cooccurrence of mutated and wild type copies of mtDNAs is named heteroplasmy. Many studies –83 showeIn humans most of the microorganisms reside in the intestinal tract and their role is so central that all these microbes are considered as an additional organ, characterized by its own genome . The gutThe composition of the microbiota strongly impacts on the host health. Indeed, several studies report that the dysbiosis of the microbiome occurs in different chronic conditions, including obesity, inflammatory bowel diseases, and diabetes –96.Regardless of whether it is a cause or a consequence of diseases, the GM can actively contribute to diseases consolidation. Indeed, several studies on disorders show that a pathologic phenotype can be transmitted from a diseased animal to a healthy recipient through the graft of the microbiota and this also applies to complex diseases where host genetics and environmental factors play a role , 98. The Bacteroidetes proportion remains unchanged in elderly, whereas in a study on Irish elderly [ Bacteroidetes strikingly increase and overcome the Firmicutes. The influence of different cultural habits and lifestyle in the two populations considered is likely the major force underlying the observed differences [The age-related changes in gut microbiota are very controversial and results from recent studies are not always concordant. In fact the study by Biagi et al. , perform elderly  Bacteroiferences , even ifferences . This obferences  and by aferences , 105. Inferences , supportferences . Recentlferences —and an oferences .Human physiology undergoes profound changes from birth to old age. In the elderly, these changes are mainly the result of adaptive strategies at the molecular and cellular levels to compensate the damage accumulation that occurs over time. A major contribution to these changes is played by the cell microenvironment. A growing body of evidence supports the idea that the systemic environment is the repository of danger signal products and of the whole garbage that the senescent cells and the impaired tissues produce. Several studies provide evidence of rejuvenation of aged cells by exposure to a young systemic environment, suggesting that it is the microenvironment to which the cells are exposed to that causes/maintains the old phenotype –112. TheMore than 90% of the factors required for mitochondrial function are encoded by the nuclear genome. Coevolution of mtDNA and nDNA is a crucial process that preserves biological functionality and cell activities . This is Drosophila showing that epistatic interactions can cause incompatibilities that decrease fitness [ Drosophila, the cooccurrence of naturally occurring mutations in a mitochondrial tRNA and in a nuclear-encoded tRNA synthetase that show little effect on their own severely compromises development and reproduction. The effect of this interaction affects mitochondrial functionality, supporting the hypothesis that the variable penetrance of mitochondrial DNA mutation is likely affected by mitochondrial-nuclear interactions [Similar evidences come from studies on fitness . In partractions .Interactions between nDNA and mtDNA products are possible at many levels such as protein-protein interactions in the OXPHOS, protein-RNA interactions in the mitochondrial ribosome, or nuclear factors-mtDNA recognition sites interactions in transcription and replication processes , 118. Thγ). Nevertheless, nDNA encodes not only regulatory factors, but also structural proteins that constitute the respiratory multimeric protein complexes, as well as some noncoding RNAs that are imported subsequently into the mitochondria. Complex I contains 7 mtDNA gene products and at least 25 nDNA gene products, complex II contains no mtDNA gene products and 4 nDNA gene products, complex III is made of 1 mtDNA gene product and 10 nDNA gene products, complex IV contains 3 mtDNA gene products and 10 nDNA gene products, and complex V contains 2 mtDNA gene products and 11 nDNA gene products [Regarding the first type of communication (from the nucleus to the mitochondria), the nDNA encodes for all potential factors that regulate mtDNA replication, transcription, and processing, including mtDNA polymerase  impairment) produce a signal that is transmitted from the mitochondria to the nucleus. Then, these cells produce other extracellular signals  that are able to induce a stress response  in the intestine, without altering the ETC functionality in intestinal cells. As it was observed that this mechanism extends lifespan, the authors speculate that a diffusible molecule released from one tissue might spread a sort of “longevity signal” to other tissues [Communication from the mitochondria to the nucleus involves metabolic signals (including ROS) but this field of research suffers from the lack of human studies. However, recent studies in tissues , 122. PARK2, a cytosolic E3 ubiquitin-protein ligase recruited for proteasomal-mediated degradation of outer mitochondrial membrane, significantly affect the interaction with TOMM70A and TOMM40. These results let us hypothesize that also SNPs located in genes of the TOMM machinery could be involved in the effectiveness of autophagic mitochondrial clearance. From this point of view, rs2075650 in TOMM40 that is associated to longevity, as previously described, could affect the relative configuration of the partners of TOMM complex, resulting in a more efficient clearance of damaged mitochondria. In this context, further studies are needed to elucidate the interactions between mtDNA and nuclear gene variants assessing their association with longevity in human.The mechanisms that regulate the communication between these two genomes are not completely elucidated. To date, it is well known that the balance of the crosstalk between the nDNA and the mtDNA is essential for cellular homeostasis and events that perturb this delicate equilibrium increase the vulnerability of the cell and, thereby, the rate of ageing. Regarding the ageing process, a particular attention should be devoted to SNPs located in nDNA or in mtDNA that may affect the communication between these genomes. An interesting papers by Bertolin et al.  focuses The study of epistatic interactions represents a challenge  that is The three-way interaction between human genetics, environment, and microbiota fundamentally shaped the biological history of modern human populations and continues to affect healthy globally. The disruption of this interaction and stability by modifying one or more of these three components may be a trigger for the development of diseases.A recent paper tried to determine to what extent the gut microbiota is determined by the host or by environmental factors, such as diet. This study demonstrated that the composition of gut microbiota among great ape species is phylogenetically conserved and pattern of relationship inferred from the analysis of the microbial communities was very similar to that inferred from host mitochondrial DNA, suggesting that host genome is a fundamental factor in determining microbial composition . Ley andThe role of human genetics in the assembly of the human intestinal microbiota is still controversial. Turnbaugh et al.  reportedA recent study in mice investigated how environmental stimuli and host genetic factors combine together to shape microbiota composition. In particular, this is the first study that tries to estimate the effects of maternal environment and host genetics. The results showed 18 host quantitative trait loci (QTL) in linkage with specific microbial taxa. In particular some of these loci affect species composition, others affect taxa, and others exert a pleiotropic effect. The authors suggest that the core measurable microbiota can be used in GWASs in humans .Moreover, there is a clear evidence that host-microbe crosstalk involves the immune system, and in particular the gut-associated lymphoid tissue (GALT) likely developed to oversee the interaction between bacteria that live close to the intestinal mucosal surfaces . In thisFrom an evolutionary perspective, the GALT-GM immunological crosstalk and the related tolerance of the immune system to the gut microbes have been a basal trait in mammalian evolution. Thus, the coevolutionary trajectories between mammals and their GM have been shaped by adaptive changes in the host genes that play a primary role in the crosstalk with the harbored microbial communities.MEFV) gene encodes a protein called pyrin/marenostrin that controls inflammation by interacting with the cytoskeleton in white blood cells and that, when mutated, leads to an autoinflammatory disorder called “familial Mediterranean fever” [ MEFV gene are associated with a shift in the Bacteroidetes, Firmicutes, and Proteobacteria phyla and that patients with familial Mediterranean fever have a lower microbial diversity. MYD88 gene is involved in communications between sensors of microbial products during inflammatory responses and loss of MYD88 compromises the innate immune response to pathogens [ MYD88-deficient and wild-type mice was compared and it was found that three bacterial families  differ in their relative abundance [ Candidatus Savagella [ APOA1 gene have been associated with the risk of cardiovascular diseases [ APOA1-deficient mice has a different community structure in comparison with that of wild-type mice [GWASs identified genes of both innate and adaptive immunity as relevant for inflammatory diseases, and interestingly several of these genes have been shown to have a role in shaping the gut microbial community. Genetic variants of several nuclear genes for components of immune system showed to have an impact on the composition of the gut microbiome. Mediterranean Fever (n fever” . Khachatn fever”  demonstrathogens . The micbundance . Studiesavagella ; furtheravagella . Other gdiseases  and withdiseases . Zhang eype mice .However, despite this clear coevolution between gut microbiota and host genomes, external stimuli are likely to play a crucial role in determining interindividual variability . Diet isThis overview clearly shows that the knowledge of the genetics of human ageing relies on three pillars, that is, nuclear genetics, mitochondria genetics, and microbiome genetics. Until now this research field has suffered the fact that these three genetic domains have been studied separately, and it is urgent to set up investigations capable of analyzing these three genomics altogether. If these considerations are correct, the phenotypic characterization of the people/models used in genetic studies of ageing and longevity becomes critical, particularly regarding the lack of information of GM in most of the studies. The poor knowledge of GM genetics is critical because it is closely related to anthropological variables such as nutrition and diet. Similar considerations must be applied to the fact that mitochondrial genetics is not considered in GWASs, as the mitochondrial genome is closely related to human population evolution that is in turn molded by environmental adaptation to climate and diet. Finally, all the three genetic components of longevity change with age, and this is particularly evident in GM and mtDNA but it also applies to more subtle, but quantitatively more important, mutations in nDNA. Overall, the emerging scenario is that of a continuous and dynamic interaction between the different components of the genetics of human longevity that was unpredictable until recently and is currently largely unexplored.


Transfemoral approach (TFA) remains the most common vascular access for percutaneous coronary intervention (PCI) in many countries. However, in the last years several randomized trials compared transradial approach (TRA) with TFA in patients with acute coronary syndrome (ACS), but only few studies were powered to estimate rare events. The aim of the current study was to clarify whether TRA is superior to TFA approach in patients with ACS undergoing percutaneous coronary intervention. A meta-analysis, meta-regression and trial sequential analysis of safety and efficacy of TRA in ACS setting was performed.Medline, the Cochrane Library, Scopus, scientific session abstracts and relevant websites were searched. Data concerning the study design, patient characteristics, risk of bias, and outcomes were extracted. The primary endpoint was death. Secondary endpoints were: major bleeding and vascular complications. Outcomes were assessed within 30 days. Eleven randomized trials involving 9,202 patients were included. Compared with TFA, TRA significantly reduced the risk of death , but this finding was not confirmed in trial sequential analysis, indicating that sufficient evidence had not been yet reached. Furthermore, TRA compared with TFA reduced the risk of major bleeding  and vascular complications ; these findings were supported by trial sequential analyses.In patients with ACS undergoing PCI, a lower risk of death was observed with TRA. Nevertheless, the association between mortality and TRA in ACS setting should be interpreted with caution because it is based on insufficient evidence. However, because of the clinical relevance associated with major bleeding and vascular complications reduction, TRA should be recommended as first-choice vascular access in patients with ACS undergoing cardiac catheterization. Percutaneous coronary intervention (PCI) represents a cornerstone for the treatment of patients with acute coronary syndrome (ACS). Currently, transfemoral approach (TFA) is the most common access for PCI in many countries www.acc.org, www.americanheart.org, www.europcronline.com, www.escardio.org, www.clinicaltrialresults.org, www.tctmd.com and www.theheart.org). The reference list of relevant studies was additionally scanned. No language, publication date, or publication status restrictions were imposed. The last search was run on 15th June, 2013. The following search terms were matched: “femoral”, “radial”, “transradial”, “transfemoral”, “percutaneous coronary intervention”, “randomized”, “acute coronary syndrome”, “myocardial infarction”, “unstable angina”, “non-ST-segment elevation”, “ST-segment elevation”.We searched Medline, the Cochrane Library, Scopus, scientific session abstracts , and relevant websites  random treatment allocation; 2) inclusion of patients with ACS; and 3) the use of TRA in the experimental arm. Exclusion criteria were: 1) ongoing studies; 2) irretrievable data and 3) trials not reporting death occurrence during follow-up. Complete electronic search strategy for Medline (PubMed) and The Cochrane Library was reported in the Supporting Information.Two investigators (R.P. and G.G.) independently assessed reports for eligibility at title and/or at abstract level, with divergences resolved with a third reviewer (F.P.); studies that met inclusion criteria were selected for further analysis. The risk of bias was evaluated by the same two reviewer authors, in accordance with The Cochrane Collaboration methods and considering the following methodological items: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, other bias and sample size calculation. We did not use a quality score, since this practice has been previously discouraged The primary endpoint of this meta-analysis was death within 30 days. Secondary endpoints were: major bleeding and vascular complications. Per protocol definitions of clinical endpoints were reported in the 2 statistic, which describes the percentage variation across studies that is due to heterogeneity rather than chance. As a guide, I2 values <25% indicated low, 25–50% indicated moderate, and >50% indicated high heterogeneity metareg command, as previously described Statistical analysis was performed with STATA 11 statistical software . The к statistic was used to assess agreement between reviewers for study selection. Odds ratio (OR) and 95% confidence intervals (95% CI) were used as summary statistics. The pooled OR was calculated by using the fixed effects Mantel-Hænzel model, while, in case of significant heterogeneity across studies, the random effects DerSimonian and Laird model was reported instead. In case of statistical significance, the number needed to treat (NNT) and the number of avoided events per 1,000 treated patients were provided. The Breslow-Day chi-squared test was calculated to test the statistical evidence of heterogeneity across the studies (p<0.1). In addition, we used the Iwww.ctu.dk/tsa). This is a methodology that combines an a priori information size calculation for a meta-analysis with the adaptation of monitoring boundaries to evaluate the accumulating evidence Trial sequential analysis was performed according to the monitoring boundaries approach The study was realized in compliance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement As reported in 2 = 0%; 95% CI, 0–65%; phet = 0.94). Visual inspection of funnel plot did not reveal a skewed distribution for death, suggesting the absence of small-study effects . As reported in  effects . MoreoveTrial sequential analysis showed a lack of sufficient evidence of a benefit of TRA for the reduction of death. Only the 33%  of the required sample size was accrued to detect a 20% relative risk reduction for death .2 = 0%; 95% CI, 0–65%; phet = 0.83). The NNT to prevent one major bleeding with TRA was 160.8 and 6.2  major bleedings were prevented in each 1,000 patients treated; these data were based on an OR = 0.60 applied to the control group event rate.Major bleeding was reported in a total of 116 patients (1.25%). As shown in In trial sequential analysis, despite the required information size was not met , the cumulative Z-curve crossed the trial sequential monitoring boundary, indicating that sufficient evidence exists for a 35% reduction in the relative risk of major bleeding with TRA .2 = 0%; 95% CI, 0–62%; phet = 0.74). The NNT to prevent one vascular complication with TRA was 30.8 and 32.4  vascular complications were prevented in each 1,000 patients treated; these data were based on an OR = 0.35 applied to the control group event rate.Data about vascular complications were available for 9,053 patients (98%). A total of 318 patients (3.51%) had vascular complications. As reported in Trial sequential analysis confirmed that TRA is superior to TFA in reducing vascular complications .Influence analysis demonstrated that no single study significantly altered the summary ORs for the endpoints, because one at a time study omission did not result in a movement of the point estimate outside the 95% CI .None of the study-level covariates significantly influenced the risk of study endpoints at meta-regression analysis . FurtherThe main findings of this study are that: TRA compared with TFA reduced the risk of major bleeding and vascular complications in patients with ACS; despite the meta-analysis showed a reduced risk of death with TRA, there is no definite evidence supporting this association as demonstrated by trial sequential analysis; the meta-regression analysis suggested that the benefit with TRA, in terms of bleeding and vascular complications, was irrespective of the patient risk profile.The incidence of recurrent ischemic events in patients with ACS has been drastically reduced by the combination of multiple antithrombotic agents, along with the early use of coronary angiography with a view to revascularization Despite we found a 30% reduction in the odds of death in favour of TRA, with about 7 deaths prevented per 1,000 patients treated, this finding was not confirmed in trial sequential analysis, which disclosed an insufficient evidence for this association. Indeed, current available data do not conclusively support a 20% relative risk reduction in mortality with TRA, since only one third of the required study population was accrued in this study. Thus, trial sequential analysis should be implemented when a meta-analysis is performed, since many apparently conclusive meta-analyses may become inconclusive when the statistical analyses take into account the risk of random error due to repetitive testing.In contrast with previous meta-analyses The decreased risk of major bleeding, along with the dramatic reduction in vascular complications, might provide an explanation to the possible risk reduction in death. In this respect, a large pooled analysis of three randomized trials including ACS patients found that approximately 1 in 10 patients who developed major bleeding died during the first 30 days after hospitalization compared with 1 in 40 of those who did not develop major bleeding Meta-regression analysis showed consistent results in single- versus multi-centre trials. This was an important finding since single-centre trials are usually associated with a larger treatment effects than multi-centre trials First, this is a meta-analysis at the study level and we could not properly assess the role of confounding factors. Second, all included trials were performed by experienced operators skilled in TRA, thus limiting the external validity of the results of this meta-analysis for centres mainly performing transfemoral procedures. This reinforces the need for dedicated training programs for TRA, especially in teaching hospitals This study demonstrated that the use of TRA in patients with ACS undergoing invasive management was associated with a significant reduction in the risk of major bleeding and vascular complications, as compared with TFA. The robustness of these findings was confirmed by trial sequential analysis. The clinical benefit, in terms of major bleeding or vascular complications, might translate in a lower risk of death with TRA. Nevertheless, the association between mortality and TRA in ACS setting should be interpreted cautiously because it is based on insufficient evidence, as demonstrated by trial sequential analysis.However, because of the clinical relevance associated with major bleeding and vascular complications reduction, TRA should be recommended as first-choice vascular access in patients with ACS undergoing cardiac catheterization.File S1(DOC)Click here for additional data file.Checklist S1(DOC)Click here for additional data file.