MTHFR gene. Several studies have assessed the association between MTHFR C677T polymorphism and the risk of congenital heart defects (CHDs), while the results were inconsistent.Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme for folate metabolism in humans; it is encoded by the MTHFR C677T polymorphism and CHDs risk in children with heterogeneity (Pheterogeneity = 0.000) and publication bias (Pegger = 0.039), but it turned into null after the trim-and-fill method was implemented . Nevertheless, positive results were obtained after stratified by ethnicity and sample size in all subgroups except the mixed population. For mothers, there was significant association between the variant and CHDs without heterogeneity  and publication bias (Pegger = 0.981). However, the results varied across each subgroup in the stratified analysis of ethnicity and sample size.Multiple electronic databases were searched to identify relevant studies published up to July 22, 2012. Data from case-control and TDT studies were integrated in an allelic model using the Catmap and Metafor software. Twenty-nine publications were included in this meta-analysis. The overall meta-analysis showed significant association between MTHFR C677T polymorphisms may contribute to the risk of CHDs.Both infant and maternal  Congenital heart defects (CHDs) refer to the defects in the structure of the heart and great vessels present at birth. The incidence of CHDs is reported to be 5–8‰, making them one of the most common congenital disorders in newborns. Besides, they are the leading causes of infant death worldwide, with a mortality of 24.1% MTHFR gene is located at 1p36.3 of humans. The missense mutation, C677T (rs1801133), is one of the most investigated single nucleotide polymorphisms (SNPs) MTHFR with reduced enzymatic action, which causes increased plasma homocysteine concentrations Methylenetetrahydrofolate reductase (MTHFR), as a crucial enzyme for the metabolism of folate, plays an important role in processing amino acids and building blocks of proteins. Specifically, it converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. This reaction is a multistep process, in which, MTHFR converts the homocysteine to another amino acid, methionine. Then, the methionine was used by body to make proteins and other important compounds MTHFR 677TT genotype was a risk factor for CHDs in a case-control study of 114 Causasian patients and 228 healthy controls. After this report, a string of studies have subsequently explored the association between this variant and risk of CHDs, however the results were controversial. Besides, single study may have limited statistical power to detect the modest effect of MTHFR C677T polymorphism on CHDs. Therefore, we conducted a meta-analysis of the available data and used the method described by Kazeem et al. MTHFR C677T polymorphism in CHDs.Junker and colleagues firstly proposed that infant We designed and reported the meta-analysis according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement (Checklist S1).MTHFR” and “polymorphism or variant”. To expand the coverage of our searches, we further carried out searches in Chinese National Knowledge Infrastructure (CNKI) and Wanfang databases with the translation of all English searching terms. Reference lists of the retrieved articles were also scanned for more eligible studies. We included case-control studies and TDT with human subjects that investigated the association between MTHFR C677T polymorphism and CHDs risk in all languages. All phenotypes of CHDs, such as patent formen ovale, atrial septal defect, patent ductus arteriosus, coarctation of the aorta, were included in this meta-analysis. Animal studies, reviews, simply commentaries, case reports and unpublished reports were excluded. Additionally, studies with overlapping data were carefully examined, and the study that included the largest number of subjects was finally selected.We conducted an electronic search for relevant articles published before July 22, 2012 in PubMed, EMBASE, and ISI Web of Science databases with the combination of the following terms: “congenital cardiac/heart defects”, “methylenetetrahydrofolate reductase or All data were extracted independently by two authors (W. Wang and YJ. Wang), and any disagreement was adjudicated by the corresponding author. The following information was extracted or calculated from each study: first author, year of publication, country of origin, ethnicity, type of CHDs, study design, number of cases and controls, counts of alleles in case and control groups in case-control studies and numbers of transmitted alleles from heterozygous parents to affected offspring in family-based studies. If there were multiple ethnic populations included in a study, each population was considered separately.2χ test. Data from case-control and TDT studies were summarized in two-by-two and two-by-one tables respectively. Odds ratios (ORs) and their 95% confidence intervals (CIs) were recalculated based on allele data using the method reported by Kazeem et al. 2χ-based Cochran’ Q-test, in which heterogeneity was considered significant at P<0.1. Pooled OR was calculated by the fixed-effect model when heterogeneity was negligible, whereas, the random-effect model was adopted. An allelic model was assumed for MTHFR C677T to combine results of case-control or TDT studies. For the synthesis of case-control and TDT studies, the method described by Kazeem et al. P values were two-sided with a significant level at 0.05 except for the one in Q test for heterogeneity, and all analyses were carried out with Catmap and Metafor software in R program (version 2.9.2).Hardy-Weinbery equilibrium (HWE) in controls was assessed again in each included studies by the goodness-of-fit MTHFR C677T polymorphism and CHDs (4 studies), without case-control or TDT design (11 studies), data duplication (6 studies) and lack of sufficient data (7 studies). Nevertheless, 6 publications were appended by scan of the references of the retrieved articles. Thus, 29 publications involving 2554 cases and 3838 controls in 20 case-control studies for children, 1837 cases and 16351 controls in 14 case-control studies for mothers, and 572 families in 2 TDT studies were ultimately included MTHFR C677T polymorphism in children, significant heterogeneity was detected (Pheterogeneity = 0.000), thus the random-effects model was employed. Compared to the C allele, the T allele conferred a pooled OR of 1.30 (95% CI = 1.13–1.49) in allelic model (Pegger = 0.039), therefore, the trim-and-fill method under random-effects model was implemented. However, no significant association between MTHFR C677T polymorphism and CHDs risk  was found after 6 virtual studies were appended.Among the 22 studies of the ic model . Becauseic model . As reflic model  and EggePheterogeneity in all subgroups < 0.10). In the Asian or European population, the allelic model exhibited significant association with CHDs risk , whereas there was no positive reslut in the mixed population . Then the data were further stratified by sample size into larger-sample-size and small-sample-size subgroups (Pheterogeneity = 0.000), but not in small-sample-size subgroup (Pheterogeneity = 0.250). Nevertheless, significant associations between MTHFR C677T polymorphism and the risk of CHDs were observed in both subgroups . The stratified analysis by study design could not be performed due to only 2 TDT studies obtained.To investigate the potential source of heterogeneity, stratified analysis was initially performed by ethnicity . After subgroups . SignifiMTHFR C677T polymorphism in mothers (Pheterogeneity = 0.150), thus the fixed-effects model was adopted. Significant association between the variant and CHDs was detected, with the T allele conferring a pooled OR of 1.16 (95% CI = 1.05–1.29) (Pegger = 0.981).There was no evidence of heterogeneity among 14 studies of the 05–1.29) . The sen05–1.29) . Besides05–1.29)  or the EMTHFR C677T polymorphism with the risk of CHDs in different populations, stratified analysis by ethnicity was conducted (Pheterogeneity = 0.750) or European (Pheterogeneity = 0.334) population, whereas the heterogeneity appeared in the mixed population (Pheterogeneity = 0.028). The polymorphism presented significantly increased risk of CHDs in the Asian population , but not in the European  or mixed  population. The data were additionally stratified into larger-sample-size and small-sample-size subgroups . However, with regard to the small-sample-size subgroup, there was no evidence of association in the random-effects model . The pooled OR was not appraised in the case-control and TDT studies separately because of no study with TDT design.To explore the association of onducted . No signubgroups . In the MTHFR C677T polymorphism and CHDs risk in children with publication bias, however it became non-significant after we applied the trim-and-fill method to address the problem of publication bias. Nevertheless, positive results were detected after stratified by ethnicity and sample size in all subgroups except the mixed population. With respective to mothers, there was significant association between the polymorphism and CHDs without heterogeneity, whereas the results differed from each subgroup in the stratified analysis of ethnicity and sample size. To the best of our knowledge, this current meta-analysis firstly integrated the case-control and TDT studies to reflect the precision effect of MTHFR C677T polymorphism in CHDs risk.The overall meta-analysis comprising 29 case-control and TDT studies showed significant association between MTHFR C677T polymorphism and the risk of CHDs were found in all subgroups barring the mixed population, thus we conjectured that the null association might result from the mixed genetic backgrounds. For stratified analysis by ethnicity, the heterogeneity was still detected in all subgroups, suggesting the ethnicity might not be a source of heterogeneity. With respect to the stratified analysis by sample size, heterogeneity disappeared in small-sample-size subgroup but still existed in the other group, which was probably explained by relatively weaker heterogeneity on subjects’ characteristics and genotyping methods across studies with small sample size. For mothers, the MTHFR C677T polymorphism presented significantly elevated risk of CHDs without heterogeneity and publication bias. Moreover, the sensitivity analysis further indicated the stability of our results. In the stratified analysis by ethnicity, no obvious heterogeneity in the European and Asian populations was observed, whereas it remained in the mixed population, which was similar with the results in children. However, significant association was merely found in the Asian population, implying there might be different modes of action for this polymorphism across ethnicities. As far as stratified analysis of sample size was concerned, positive results were detected in the large-sample-size subgroup but not in the small-sample-size, implying the null association was probably caused by limited sample size.Although the overall meta-analysis demonstrated negative result after adjustment for publication bias, associations between infant MTHFR C677T polymorphism and CHDs risk observed in current meta-analysis was supported by multiple studies MTHFR 677TT in children was a risk factor for CHDs MTHFR 677TT and the presence of CHDs in their offspring in the study by Balderrábano-Saucedo et al. MTHFR C677T polymorphism was just associated with a specific phenotype of CHDs MTHFR C677T polymorphism and the risk of CHDs was detected in our study, but not in the meta-analysis by Yin et al. The significant association between infant and maternal MTHFR C677T polymorphism may influence the occurrence of CHDs through homocysteine. As a free amino acid, homocysteine exists in either the reduced  or oxidized  form. Thiols can initiate lipid peroxidation, produce hydroxyl radical, and oxidatively cleave proteins. Therefore, regulating the oxidation state of sulfur-containing amino acids is an important strategy for curbing cellular damage Compared with meta-analyses in 2012 MTHFR C677T polymorphisms may be significantly associated with the risk of CHDs. But more studies with large sample sizes are required to confirm current findings and explore the interaction between genetic polymorphism, folic acid intake and CHDs risk.In conclusion, our meta-analysis demonstrated that both infant and maternal Figure S1The funnel plots of the MTHFR C667T in children for CHDs.(TIFF)Click here for additional data file.Figure S2The funnel plots of the MTHFR C667T in mothers for CHDs.(TIFF)Click here for additional data file.Table S1Sensitivity analysis of pooled OR for MTHFR C667T polymorphism in children.(DOCX)Click here for additional data file.Table S2Sensitivity analysis of pooled OR for MTHFR C667T polymorphism in mothers.(DOCX)Click here for additional data file.Checklist S1PRISMA checklist.(DOCX)Click here for additional data file.


Myeloperoxidase (MPO) is an endogenous oxidant enzyme that produces reactive oxygen species (ROS) and may be involved in lung carcinogenesis. The MPO−463G>A polymorphism influences MPO transcription and has been associated with lung cancer susceptibility. However, the association between the MPO−463G>A polymorphism and lung cancer risk remains controversial.Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of the association.To investigate the effect of this polymorphism on lung cancer susceptibility, we performed a meta-analysis based on 22 published case–control studies including 7,520 patients with lung cancer and 8,600 controls. Overall, there was no evidence for significant association between MPO−463G>A polymorphism and lung cancer susceptibility . In the stratified analyses by ethnicity, source of controls and smoking status, we also did not find any significant association between them.In summary, this meta-analysis suggests MPO−463G>A polymorphism may not be a risk factor for developing lung cancer. However, further prospective well-designed population-based studies with larger sample size are expected to validate the results. Lung cancer is one of the most common malignancies and the leading cause of cancer-related deaths in the United States and worldwide. It is estimated that in 2011, approximately 221,000 new cases will be diagnosed and 156,900 deaths due to lung cancer will occur in the United States Myeloperoxidase (MPO) as an endogenous oxidant lysosomal enzyme available in polymorphonuclear neutrophils and monocytes catalyzes the reaction between the chloride ion and hydrogen peroxide and generates hypochlorous acid and other reactive oxygen species (ROS) http://www.ncbi.nlm.nih.gov/projects/SNP). The most extensively studied single nucleotide polymorphism (SNP) was −463G>A polymorphism (rs2333227) located in the promoter region of the MPO gene. Since Austin et al first published the MPO−463G>A polymorphism in 1993 The human MPO gene located on chromosome 17q23.1 consists of 12 exons and 11 introns So it is necessary and significant to perform a meta-analysis to explore the association between the MPO−463G>A polymorphism and lung cancer risk. Therefore, we performed a meta-analysis on all eligible case–control studies to estimate the overall lung cancer risk of MPO−463G>A polymorphism and to quantify heterogeneity among the individual eligible studies.Eligible literatures published before the end of September 2012 were identified by the search of PUBMED, EMBASE, ISI Web of Science databases using combinations of the following keywords: “Myeloperoxidase”, “MPO”, “polymorphism” or “variant” and “lung cancer” or “lung carcinoma” without restriction on language. All relevant publications were reviewed. Articles in reference lists were also hand-searched for potentially relevant publications.Studies included had to meet all the following inclusion criteria: (a) the diagnosis of lung cancer patients were confirmed histologically or pathologically; (b) a case–control study on MPO−463G>A polymorphism and lung cancer; (c) sufficient available data for estimating an odds ratios (ORs) with 95% confidence intervals (CIs). Major reasons for exclusion of studies were as follows: (i) not for lung cancer study, (ii) only case population and (iii) duplicate of previous publication.Two investigators  extracted the data independently including first author, year of publication, country, ethnicity , source of controls , genotyping methods, matching variables, number of genotypes in cases and controls. Discrepancies were adjudicated by the third investigator (YF Zhou) until consensus was achieved on every item. Because two studies P<0.10 was considered significant) P<0.05 was considered significant. All statistical analyses were performed with STATA software , and all tests were two sided.For control group of each study, the allelic frequency was calculated and the observed genotype frequencies of the MPO−463G>A polymorphism were assessed for Hardy-Weinberg equilibrium (HWE) by using the chi-square test. We calculated the strength of the association between MPO−463G>A polymorphism and lung cancer risk by odds ratios (ORs) corresponding to 95% confidence intervals (CIs). The pooled ORs were performed for homozygote model (GA versus GG), heterozygote model (AA versus GG), dominant model (AA/GA versus GG) and recessive model (AA versus GA/GG) respectively. Stratified analyses were also performed by ethnicity, source of controls and smoking status respectively. Heterogeneity was evaluated with a chi-square-based Q test  Table 3.Sensitivity analysis was performed through sequentially excluding individual studies. Statistically similar results were obtained after sequentially excluding each study in GA/AA versus GG model Figure 4P = 0.55), GA versus GG (P = 0.27), AA/GA versus GG (P = 0.56) and for AA versus GA/GG (P = 0.61).The funnel plot and Egger’s test did not provide any obvious evidence of publication bias that examined the MPO−463G>A polymorphism and lung cancer risk. The shape of funnel plots did not reveal any evidence of funnel plot asymmetry Figure 5As we all know, lung cancer is a complex multifactorial and multistage process, in which both host genetic factors and environmental factors are involved Oxidative stress occurs when the excessive production of reactive oxygen species (ROS) overwhelms the antioxidant defense system. Increasing evidences suggests variability in these genes involved in oxidative stress may determine the level of oxidative stress in the organism and play a crucial role in carcinogenesis Population stratification is an area of concern that can lead to spurious evidence for the association between a marker and cancer and suggest a possible role for ethnic differences in genetic backgrounds.Previous studies have found a wide variation in the A allele frequency of the MPO−463G>A polymorphism across different ethnicities. The −463A allele frequency was 22.8% in European population, but approximately 14.7% in Asian population Lung cancer have been characterized as causally related to cigarette smoking Several limitations of this meta-analysis should be addressed. First, when interpreting the results of this meta-analysis, heterogeneity was a potential problem and the origins of heterogeneity may include many factors, such as the differences in control characteristics and diverse genotyping methods; Second, the lack of detailed information such as age, sex and lifestyle of the patients in some studies, limited further stratification, and more accurate ORs would be corrected for age, sex and other factors that were associated with lung cancer risk. Nevertheless, our meta-analysis has some advantages. First, the well-designed search and selection method significantly increased the statistical power of this meta-analysis. Second, we could perform a subgroup analysis to address a possible interaction between smoking parameters and MPO−463G>A polymorphism. Third, the results did not show any evidence of publication bias.In summary, our meta-analysis suggests that the MPO −463G>A polymorphism may not be a risk factor for lung cancer. However, lung cancer may be a multifactorial disease that resulted from complex interactions between genetic and environmental factors, we could not collect the detailed original data of MPO gene polymorphisms and then we were not able to investigate potential gene-gene, gene- environment interactions. Further prospective researches using adjusted individual data with large sample studies which study the relationship between MPO−463G>A polymorphism and the risk of lung cancer are necessary and expected, which would lead a better, comprehensive understanding of the association between MPO−463G>A polymorphism and lung cancer risk.Checklist S1MOOSE Checklist.(DOC)Click here for additional data file.


The present study was designed to explore the association of angiotensin converting enzyme (ACE) gene insertion/deletion  polymorphism, plasma ACE activity, and circulating ACE mRNA expression with essential hypertension (EH) in a Chinese population. In addition, a new detection method for circulating ACE mRNA expression was explored.The research was approved by the ethics committee of Zhejiang Provincial Center for Disease Prevention and Control. Written informed consent was obtained prior to the investigation. 221 hypertensives (cases) and 221 normotensives (controls) were interviewed, subjected to a physical examination, and provided blood for biochemical and genetic tests. The ACE mRNA expression was analyzed by real time fluorescent quantitative Reverse Transcription PCR (FQ-RT-PCR). We performed logistic regression to assess associations of ACE I/D genotypes, ACE activity, and ACE mRNA expression levels with hypertension.The results of the multivariate logistic regression analysis showed that the additive model  of the ACE genotype revealed an association with hypertension with adjusted OR of 1.43(95% CI: 1.04-1.97), and ACE ID genotype with adjusted OR of 1.72(95% CI: 1.01-2.92), DD genotype with adjusted OR of 1.94(95% CI: 1.01-3.73), respectively. In addition, our data also indicate that plasma ACE activity (adjusted OR was 1.13(95% CI: 1.08-1.18)) was significantly related to hypertension. However, the plasma ACE mRNA expressions were not different between the cases and controls.ACE I/D polymorphism and ACE activity revealed significant influence on hypertension, while circulating ACE mRNA expression was not important factors associated with hypertension in this Chinese population. The detection of circulating ACE mRNA expression by FQ-RT-PCR might be a useful method for early screening and monitoring of EH. Essential hypertension (EH) is a major public health burden worldwide and has been steadily increasing for the past several years in China. According to the Chinese National Nutrition and Health Survey in 2002, the prevalence of EH among adults (over 18 years old) was found to be 18.8% in China. EH is a multifactorial complex disorder caused both by genetic and environmental factors. Approximately 30% of the interindividual variability in blood pressure is estimated to be genetically determined. Numerous studies have been focused on the role of genetic variation in genes implicated in the renin-angiotensin system (RAS), particularly the angiotensin-converting enzyme (ACE) gene.RAS is one of the most important systems in blood pressure regulation. ACE is the core enzyme in the RAS catalyzing the production of angiotensin II (Ang II). Ang II is the key effector in RAS which functions as a regulator of blood pressure, water-salt balance, and angiotasis. A 287 bp insertion/deletion I/D, rs4646994) polymorphism in intron 16 of the ACE gene gives rise to the homozygotes II and DD, and the heterozygote ID. Some studies have shown an association between the DD genotype and hypertension , other s646994 poThe association of the D-allele or DD genotype with significantly higher ACE mRNA expression in tissue has been clearly documented ,15. HoweThe research was approved by the ethics committee of Zhejiang Provincial Center for Disease Prevention and Control. Written informed consent was obtained prior to the investigation.Subjects came from a community baseline investigation on chronic disease in Zhejiang Province in recent years. Seven counties were selected in the investigation based on their mean income and typical socio-economic status in a multiple-stage stratified cluster randomization sampling method, then one community was selected from each above county in a simple randomization sampling rule. Several small residential areas were randomly sampled in each community and a cross-sectional study was carried out in these seven communities of the population aged 35 years and over.A total of 9485 residents were investigated. Among them 1857 residents were confirmed EH according to the diagnostic criteria of EH established by World Health Organization (WHO) / International Society of Hypertension (ISH) in 1999. Cases were selected among the 1857 EH patients if they had been untreated for 2 weeks prior to the conduct of the study and if they had no diabetes, no history of stroke, no coronary heart disease, neither gout. There are totally 221 cases who met the above criteria. The controls were 1:1 matched with the cases by small residential area, gender and age (±5 years). 221 controls were selected in healthy individuals without EH, diabetes, stroke history, coronary heart disease, or gout. Written informed consent was obtained prior to the investigation.The main instruments used including a fluorescence quantitation polymerase chain reactor , an auto-biochemical analyzer , a biosafety cabinet , and an ultraviolet Nano-drop1000 spectrophotometer .All the investigators for this study received training and passed qualifying examinations. The field investigation included administration of an epidemiological questionnaire, a physical examination, and laboratory tests.The epidemiological questionnaire obtained general demographic characteristics, family history, medical history, and other factors including smoking, drinking, physical health, diet(food frequency questionnaires), and mental health.2 (m2)Investigators performed physical examinations to obtain height, weight, waist circumference (WC), systolic blood pressure (SBP), and diastolic blood pressure (DBP) of participants. The body mass index (BMI) was calculated as follows: BMI= weight (kg) / heightParticipants were asked about their smoking status. Those who responded as abstained smoking means they smoked but abstained for more than one year. Occasionally smoking was defined as those who occasionally smokes but does not meet the frequently smoking standard. Frequently smoking was defined as smoking every day for more than one year. People who did not smoke were defined as never smokers.While people who did not drink were categorized as never drinking, abstained drinking was labeled for those who drank but abstained for more than one year. Occasionally drinking was defined as those who occasionally drinks but does not meet the frequently drinking standard. Frequently drinking was defined as consumption of alcohol every day for more than one year.Individuals with mild occupational physical activities included those who work in an environment such as an office, retail or school. Those with moderate occupational physical activities included work in construction or its equivalent in physical activity. Individuals with heavy occupational physical activities included those that work as non-mechanized farming laborers, steel workers, dancers, or equivalent. Very heavy occupational physical activities were defined as those who work as foundry labors, lumbering workers. Since the number of very heavy occupational physical activities is only 1, it was combined with heavy occupational physical activities as one group.Similarly, since the number of very high stress of work and life is also only 1, it was combined with high stress of work and life as one group.A 5mL fasting EDTA-anticoagulated venous blood sample was collected from each subject. Plasma was subsequently separated by centrifugation and subjected to automated biochemical analysis to measure levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDLC), fasting blood glucose (FBG), total protein (TP), albumin (ALB), uric acid (UA), and plasma ACE activity. The remaining blood cells were stored at -80°C for the gene detection assay.5’-CTG GAG ACC ACT CCC ATC CTT TCT-3’; reverse primer 5’-GAT GTG GCC ATC ACA TTC GTC AGA T-3’. The PCR products were analyzed on 2% agarose gels with a 100-bp DNA Ladder (Promega), and images were acquired and analyzed using a gel imaging system.QIAamp DNA Blood Mini kits  were used to extract DNA from blood cells. Primers used for the ACE I/D polymorphism analysis were as previously described : Both foThe ACE gene I/D polymorphism transmission models were considered: (1) additive , (2) dominant (DD + ID versus II), and (3) recessive (DD versus ID + II) models of the D allele.Real-time fluorescent quantitative RT-PCR was used to measure ACE mRNA expression. The sequence encoding the ACE gene was retrieved from GenBank. The real-time fluorescent quantitative RT-PCR primers and TaqMan probes used to detect the human ACE gene and the internal reference gene (β-actin) were designed using the Primer Express software (PE Applied Biosystems).5’-AGC CAA CCA CAC CCT GAA GT-3’,Forward primer: 5’-GTG TTC TGC AAC TGG TTC ACA TC-3’,Reverse primer: 5’-CGG CAC CCA GGC CAG GAA GTT-3’, labled by dye FAM.Fluorescent probe: 5’-CCG TCT TCC CCT CCA TCG-3’,Forward primer: 5’-GTC CCA GTT GGT GAC GAT GC-3’,Reverse primer: 5’-CCA GGG CGT GAT GGT GGG CA-3’, labled by dye FAM.Fluorescent probe: Total RNA from blood cells collected from the cases and controls was extracted using QIAamp RNA Blood Mini kits . The total RNA concentration was determined using an ultraviolet spectrophotometer and 1% agarose gel electrophoresis. One-Step RT-PCR Kits  were used for this analysis. The reaction was run in an MJ Option 2 fluorescence quantitation polymerase chain reactor  in a volume of 25 µL. The following protocol was used: 40 cycles of 42°C for 30 minutes, 95°C for 2 minutes, 95°C for 5 seconds, and 54°C for 35 seconds. Reactions for each RNA sample were performed in duplicate under the same conditions. No template controls were used throughout the experiment.The total RNA (concentrated in cases of low RNA concentration) from one randomly selected subject was subjected to five-fold serial dilutions . The serial dilutions served as the reference standard and were subjected to real-time fluorescent quantitative RT-PCR along with the other samples. The copy numbers of the reference standard for the MJ Option 2 fluorescence quantitation polymerase chain reactor were defined proportionally on the basis of serial dilutions  to 625, 125, 25, 5 and 1 in this experiment.T values of the serially diluted reference standards and the previously defined copy numbers were imported into Excel. The relative quantitative standard curves for the ACE and β-actin genes were set by using the base 10 logarithm of the copy number on the x-axis and the CT value on the y-axis.The CT values from each sample into the standard curves of the ACE and β-actin genes. The relative expression levels of the standardized samples were obtained by dividing the ACE gene copy numbers by the β-actin copy numbers. The standardized relative expression levels of the EH group and the control group were compared.The copy numbers of expressed ACE and β-actin in each sample were obtained by separately inputting the averages of the two Ct-tests and the means of multiple groups were compared with one-way ANOVA analysis. Relative ACE mRNA expression levels  were log-transformed to normal distribution. The χ2 test was used to compare categorical variables. The univariate logistic regression analysis was performed to assess associations with known and suspected risk factors  between the cases and controls and group variable  was used as the dependent variable . All the significant factors from the univariate regression analysis and all the professionally known risk factors were controlled in the multivariate logistic regression analyses.All data were analyzed by using SPSS 16.0 software. The continuous variables were presented as the means ± SD. Comparisons of continuous variables were performed using ~45, 45~55, 55~65, and >65 four groups, there were 26, 64, 64, 67 individuals in each group both in cases and controls.As shown in r2 of the standard curves of the ACE and β-actin genes were 0.9834 and 0.9994, respectively.Two clear bands of 28S and 18S at a brightness ratio of 2:1 (28S: 18S) in the agarose gel electrophoresis of total RNA suggested that the RNA was intact. The ratio of A260/A280 of the RNA was measured by the ultraviolet spectrophotometer as 1.9 to 2.1. The correlation coefficient A total of 32 independent variables  genotype, relative ACE mRNA, relative ACE mRNA groups, ACE activity, ACE activity groups, TC, TC groups, TG, TG groups, HDLC, HDLC groups, FBG, FBG groups, TP, TP groups, ALB, ALB groups, UA, UA groups) were analyzed using univariate regression analysis. Among the 13 risk factors, types of cooking oil, BMI, BMI groups, WC, WC groups, preference for salty foods, family history of hypertension and family history of diabetes mellitus revealed associations with hypertension. While among testing markers, relative ACE mRNA, relative ACE mRNA groups, ACE activity, ACE activity groups, TC, TC groups, TG, TG groups, FBG, and FBG groups showed significant difference between the cases and controls, Table 2Our one-way ANOVA analysis showed that both in cases and controls, subjects who carry the homozygotes II had the highest ACE activity, followed by homozygotes DD, and people carrying heterozygote ID had the lowest ACE activity. ACE activities differed significantly between II genotype and ID genotype both in cases (P<0.01) and in controls (P<0.01). Moreover, ACE activities between II genotype and DD genotype in cases differed significantly (P<0.05). For ACE mRNA expression, there were no significant differences among the II, ID and DD genotype groups.After adjusted for age(continues), gender, BMI(continues), smoking, drinking, Waist Circumference(continues), preference for salty foods, family history of EH, family history of diabetes mellitus , types of cooking oil, CHOL(continues), TG(continues), FBG(continues), relative ACE mRNA (log transformed), and ACE activity genotype) or ACE(rs4646994) genotype (for ACE activity(continues)), which were the significant factors from the univariate regression analysis and some of the professionally known risk factors, we found that the additive model  of the ACE (rs4646994) genotype revealed a significant association with hypertension with adjusted OR of 1.43(95% CI: 1.04-1.97), and ID genotype with adjusted OR of 1.72(95% CI: 1.01-2.92), DD genotype with adjusted OR of 1.94(95% CI: 1.01-3.73), respectively. In addition, ACE activity was both significantly associated with ACE I/D polymorphism and hypertension .In this study, the distribution of the genotypes of the ACE gene was in Hardy-Weinberg equilibrium, indicating that the selected samples are representative. The potential role of the I/D polymorphism and thus of differential ACE plasma activity and ACE mRNA expression in the development of hypertension is still a matter of controversy -15,18,19It has been suggested that the increase in serum ACE activity in essential hypertension is not of pathophysiological or clinical significance . Persu eThe expression levels of ACE mRNA may be influenced by multiple factors, including developmental phases, growth conditions, lesion locations, and duration and conditions of storage. Therefore, mRNA expression levels of ACE among different tissues are incomparable. We thus explored the correlation of the EH and ACE gene I/D polymorphism with mRNA expression levels of the ACE gene with fresh blood samples. The univariate regression analysis of our study showed an association between hypertension and mRNA expression level of ACE , especially in the high mRNA expression level group , indicating a potential relationship between the aberrant mRNA expression of ACE and hypertension . There aIn a conclusion, EH involves interactions between genetic and environmental factors. previous studies illustrated that different populations with varying genes, lifestyles, dietary habits, environmental exposures,as well as the potential gene-environment interactions all contribute to the expression of hypertension ,6,19,31.-△△CT relative quantification is a simple method of real-time quantitative PCR experiments for the analysis of gene expression profiles [r2 of the standard curves of the β-actin genes was 0.9994, and it thus was proved to be an ideal internal standard.There are mainly two ways to quantitate gene expression levels: absolute quantification and relative quantification. Absolute quantification is an approach calculating the copy number of the initial template with the standard curves. Despite its accuracy and reliability, the reference standard is required to create the standard curve by cloning gene fragments and constructing the in vitro transcription system, which are complicated and difficult . The claprofiles . Howeverprofiles . In addiThe quantitative detection of the mRNA expression of ACE is relatively accurate and can be reflective of the aberrant mRNA expression of ACE. Admittedly, a great deal of research on the validation and delimitation of the corresponding threshold is still lacking for the prediction of EH through the quantitative detection of the mRNA expression levels of ACE in peripheral blood. In spite of that, owing to the wide availability of peripheral blood samples and the high sensitivity, convenience, high-accuracy, and good reproducibility of the fluorescent quantitative RT-PCR method, the detection of the mRNA expression of ACE in peripheral blood by fluorescent quantitative RT-PCR can be of great significance to early screening, prevention, treatment, surveillance, and diagnosis of EH, and thus it deserves further investigation.


PAI-1 gene may influence the risk of this disease. To comprehensively address this issue, we performed a meta-analysis to evaluate the association of PAI-1 4G/5G polymorphism with diabetic retinopathy in type 2 diabetes.Mounting evidence has suggested that plasminogen activator inhibitor-1 (PAI-1) is a candidate for increased risk of diabetic retinopathy. Studies have reported that insertion/deletion polymorphism in the Data were retrieved in a systematic manner and analyzed using Review Manager and STATA Statistical Software. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of associations.Nine studies with 1, 217 cases and 1, 459 controls were included. Allelic and genotypic comparisons between cases and controls were evaluated. Overall analysis suggests a marginal association of the 4G/5G polymorphism with diabetic retinopathy . In subgroup analysis by ethnicity, we found an association among the Caucasian population . When stratified by the average duration of diabetes, patients with diabetes histories longer than 10 years have an elevated susceptibility to diabetic retinopathy than those with shorter histories . We also detected a higher risk in hospital-based studies .PAI-1 gene potentially increased the risk of diabetic retinopathy in type 2 diabetes and showed a discrepancy in different ethnicities. A higher susceptibility in patients with longer duration of diabetes (more than 10 years) indicated a gene-environment interaction in determining the risk of diabetic retinopathy.The present meta-analysis suggested that 4G/5G polymorphism in the  In addition to being involved in tissue repair and remodeling, PAI-1 plays a critical role in the regulation of intravascular fibrinolysis. In patients with type 2 diabetes (T2D), impaired fibrinolysis is involved in the pathogenesis of DR [PAI-1 gene polymorphisms and metabolic determinants, is also elevated in DR [PAI-1 variants, the most significant variation in PAI-1 expression resides in a common single-base-pair guanine insertion/deletion polymorphism (4G/5G) within the promoter region of the PAI-1 gene at nucleotide position -675 [Diabetic retinopathy (DR), the leading cause of blindness in the working population, is associated with a strong genetic predisposition, highlighted by the familial clustering of DR ,2. Severis of DR . Increasis of DR  and the is of DR ,12. PAI-ed in DR . Compareion -675 ,14. Unliion -675 .PAI-1 4G/5G and DR risk [Considering the potential influence on the individual risk for DR due to the insertion-deletion mutation of -675 4G/5G, many studies have explored the association between  DR risk -24. HowePAI-1 4G/5G polymorphism and DR risk. There was no language limitation. We also hand-searched references of original studies or review articles on this topic to identify additional studies. The following criteria were used to select the eligible studies: they must be case-control studies on the association between PAI-1 4G/5G polymorphism and DR; and they must contain detailed and correct numbers of different genotypes for estimating an odds ratio (OR) with a 95% confidence interval (CI). When several publications reported on the same population data, the largest or most complete study was chosen. As a result, nine case-control studies were included in our meta-analysis.In this meta-analysis, a comprehensive literature research of the US National Library of Medicine's PubMed database (to 1 May 2012) was conducted using research terms including 'plasminogen activator inhibitor-1', 'PAI-1', '4G/5G', 'polymorphism', 'type 2 diabetes', 'diabetic retinopathy' and the combined phrases to obtain all genetic studies on the relationship between PAI-1 genotype. Different ethnicities were categorized as Caucasian, Asian and Pima Indian. Sources of control were divided into population-based and hospital-based controls. The average duration of diabetes was separated into longer and shorter than 10 years.Two investigators independently assessed the articles for inclusion or exclusion, resolved disagreements, and attained consistency. For each eligible study, the following information was recorded: the first author's name, the year of publication, country of origin, ethnicity, total number of patients with DR and number of participants without DR (DWR) as well as the DR/DWR distribution in each PAI-1 4G/5G polymorphism and DR risk was assessed by calculating pooled ORs with 95% CIs. We evaluated the risk using the codominant model , the dominant model (4G/4G + 4G/5G versus 5G/5G) and the recessive model (4G/4G versus 4G/5G + 5G/5G). Between-study heterogeneity was evaluated by χ2-based Q-test [P < 0.10, in which case the random-effects model  and STATA Statistical Software . A two sided P-value < 0.05 was considered statistically significant.The strength of the relationship between d Q-test  and was d method ) was usel method ) was sell method . Publicaificance ). StudieIn total, nine case-control studies including 1, 217 cases and 1, 459 controls were selected in our meta-analysis. A flow chart of the literature search, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines , is showPAI-1 variation was found associated with elevated DR risk in patients with a duration of diabetes longer than 10 years . Figure The potential presence of publication bias was evaluated quantitatively by Begg's funnel plot and Egger's test. The Begg's funnel plot appeared symmetric. The Egger's test supported that there was no significant statistical evidence of publication bias for any of the four genetic models , indicating 4G/4G in PAI-1 locus as a risk factor for DR [PAI-1 carried more risk in Caucasian but not Asian participants, even though the overall effect was positive. The differences in ethnic backgrounds, lifestyle, nutrition and living environment may partly explain this discrepancy [PAI-1 4G/5G polymorphism to the population with longer duration of T2D. This subgroup analysis manifested a gene-environment interaction and highlighted the need for implementing rigorous case-selective criterion in future studies.The earliest investigation into of PAI-1  and DR rof PAI-1 ,41. Howeof PAI-1 ,18,20,21r for DR . All ther for DR -24. Our crepancy . We alsoPAI-1 polymorphism in specific cases in DR and in co-operation with other factors cannot be totally excluded.We also observed inconsistent results between hospital-based studies and population-based studies, which may be explained by the biases in hospital-based studies. Control cases in hospital-based studies may be less representative of the general population than those from population-based studies. Genes do not work in isolation; instead, complex molecular networks and cellular pathways are often involved in disease susceptibility . Taking Several potential limitations existed in our meta-analysis and our results should be interpreted with caution. First, as no correction for multiple testing was performed in this meta-analysis, false positive results may have been induced in some fraction because of the application of multiple statistical tests, which would increase the probability of type I errors. Second, our meta-analysis is based on unadjusted estimates because of a lack of original data. For example, the accurate disease time-course of individual patients was unavailable, which may potentially have affected the results where our classification criterion was according to the mean value of the diabetes duration. Third, this meta-analysis was limited by the small sample size - especially in subgroup analysis - though the Egger's test gave no publication bias . Fourth,PAI-1 polymorphism may be associated with elevated DR risk in patients with T2D, especially in the Caucasian population and in patients who have had diabetes for longer than 10 years. Future larger scale epidemiological investigation of this topic should be conducted to validate our findings.Our meta-analysis suggests that CIs: confidence intervals; DR: diabetic retinopathy; DWR: diabetes without DR; ORs: odds ratios; PAI-1: plasminogen activator inhibitor-1; PCR: polymerase chain reaction; SNP: single nucleotide polymorphism; T2D: type 2 diabetes.The authors declare that they have no competing interests.KXZ contributed to the idea and design of this study and revised the manuscript. TYZ and CP carried out the screening procedure, performed the statistical analysis and drafted the manuscript. NDL participated in the design of the study, helped performed the statistical analysis and revised the manuscript. EZ helped to improve the English language and gave some suggestions to this manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/11/1/prepub


A cleft lip with or without a cleft palate is one of the major congenital anomalies observed in newborns. This study explored the risk factors for oral clefts in Gorgan, Northern Iran.This hospital-based case-control study was performed in three hospitals in Gorgan, Northern Iran between April 2006 and December 2009. The case group contained 33 newborns with oral clefts and the control group contained 63 healthy newborns. Clinical and demographic factors, including date of birth, gender of the newborns, type of oral cleft, consanguinity of the parents, parental ethnicity, and the mother's parity, age, education and intake of folic acid were recorded for analysis. A significant association was found between parity higher than 2 and the risk of an oral cleft . According to ethnicity, the odds ratio for oral clefts was 0.87 in Turkmens compared with Sistani people  and 1.11 in native Fars people compared with Sistani people . A lack of folic acid consumption was associated with an increased risk of oral clefts but this was not statistically significant . There were no significant associations between sex , parent familial relations , mother's age and oral clefts. The results of this study indicate that higher parity is significantly associated with an increased risk of an oral cleft, while Fars ethnicity and a low intake of folic acid increased the incidence of oral clefts but not significantly.  A cleft lip with or without a cleft palate is the most common orofacial congenital anomaly among live births . The ratAs the incidence rate of oral clefts is relatively high in Northern Iran , the popThis hospital-based case-control study was performed in three hospitals  in Gorgan, which is located in the north of Iran. Ethical approval for the study was obtained from the ethics committee of Golestan University of Medical Sciences. Gorgan is the capital city of Golestan Province and the three hospitals in this study have an annual rate of more than 10,000 deliveries, which accounts for approximately 30% of deliveries in Golestan Province, Northern Iran. Golestan Province is on the south-eastern edge of the Caspian Sea. The region has a population of about 1.5 million people and covers an area of about 20,460 square kilometers. Fars, Turkman and Sistani people are the three main ethnic groups in Gorgan. Native Fars people are the predominant ethnic group in the area with the most members, the Turkman people are an ethnic group that emigrated from central Asia more than three centuries ago, and the Sistani group emigrated from southeastern Iran half a century ago.Between April 2006 and December 2009, 33 newborns with oral clefts and 63 normal newborns (control group) and their mothers were evaluated. To form the control group, for every case we selected the next one or two healthy infants that were born.A consent form was completed by the parents of all the newborn infants in the study. All live births during the investigation were examined and screened for cleft lip and cleft palate immediately after delivery by a gynecologist. The diagnosis was later confirmed by a pediatrician according to ICD10. A questionnaire addressing the relevant clinical and demographic factors for each case and control subject was completed by the pediatrician and by a nurse during an interview with the parents. The questionnaire data included birth date and gender of the infant, type of oral cleft, consanguinity of the parents, parental ethnicity, and the mother's parity, age, education and intake of folic acid. Data were collected through interviews with mothers in the immediate postpartum period, as well as by consulting the patient records of both the mothers and newborn infants. The data analysis was performed using SPSS version 16. To investigate the factors affecting the occurrence of a cleft lip and/or palate, a logistic regression model was used to measure the crude odds ratio (OR) of the occurrence of a cleft lip and/or palate for each of the independent variables. The results are expressed as an OR with 95% confidence interval (CI). The significance level was set as P<0.05.Out of 30,308 deliveries in the three hospitals in Gorgan between April 2006 and December 2009, 33 newborns with oral clefts were included in the study as cases and 63 healthy newborns as controls. The association of the evaluated risk factors with the occurrence of a cleft lip and/or palate is depicted in . The mean maternal age in the case and control group was 25.9 and 25.1 years, respectively. There was no association between maternal age and increased risk for oral clefts. Oral clefts were found to be more common in males than females, but there was no significant association between infant gender and oral clefts. A total of 33.3% of infants with a cleft lip and/or palate were the result of consanguineous marriages, whereas this rate was 31.7% in the control group. There was no association between consanguineous marriages and an increased risk of oral clefts . However, in mothers with parity greater than 2 there was a significantly increased risk of having a child with an oral cleft compared with mothers whose parity was 1 . In terms of ethnicity, the odds ratio for oral clefts in infants of Turkmen ethnicity was 0.87  and 1.11 in infants of native Fars ethnicity  compared with infants of Sistani ethnicity. A total of 63.5% of mothers in the control group and 72.7 % in the case group did not take folic acid before conceiving or during pregnancy. A lack of folic acid consumption was associated with an increased risk of oral clefts  but this was not significant. A total of 54.5% of mothers with affected newborns lived in a rural area and 45.5% in an urban area but this was not associated with an increased risk of oral clefts .In this study, oral clefts are more common in males but this result was not statistically significant indicating there is no associated between gender and oral clefts. This result is similar to other studies in Japan  and TehrAccording to our results, no association was found between maternal age and oral clefts. This result is similar to studies by Jagomagi and colleagues  and FathThere was also no association between consanguinity and oral clefts but several studies in Pakistan , Tehran,In our study folic acid consumption was not significantly associated with oral clefts. However, Van Rooij and colleagues in the Netherlands  reportedAlso, Wilcox and colleagues reported a 39% decrease in the risk of cleft lip and palate with the use of folic acid supplements adjusted for the use of multivitamins . Based on our findings, there is also no significant association between maternal education and the risk of oral clefts or between residency in a rural versus urban area and the risk of oral clefts. Our finding regarding maternal education is similar to a study by Lebby and colleagues  and is iThe limitation of this study was the sample size. We also need to maintain an accurate database for cleft registrations; systematic record keeping is essential in this area. The present study showed that ethnicity and a lack of folic acid intake was not significantly associated with an increased risk of oral clefts in infants, however high parity was significantly associated with an increase in the rate of oral clefts. Further studies with a larger sample size including all the hospitals in Golestan Province in Northern Iran are required.


Supporting health care sector decisions using time-dependent endpoints (TDEs) such as time to progression (TTP), progression-free survival (PFS), and event-free survival (EFS) remains controversial. This study estimated the quantitative relationship between median TDE and median overall survival (OS) in multiple myeloma (MM) patients.Studies  published from 1970 to 2011 were systematically searched (PubMed). The nonparametric Spearman’s rank correlation coefficient measured the association between median TDE and OS. The quantitative relationship between TDEs and OS was estimated with a two-step approach to a simultaneous Tobit model.We identified 153 studies: 230 treatment arms, 22,696 patients and mean study duration of 3.8 years. Mean of median TDEs was 22.5 months and median OS was 39.1 months. Correlation coefficients of median TTP, PFS, and EFS with median OS were 0.51 (P = 0.003), 0.75 (P < 0.0001), and 0.84 (P < 0.0001), respectively. We estimate a 2.5 month  increase in median OS for each additional month reported for median TDEs. There was no evidence that this relationship differed by type of surrogate.TDEs predict OS in MM patients; this relationship may be valuable in clinical trial design, drug comparisons, and economic evaluation. Multiple myeloma (MM) is the second most common hematologic malignancy, after non-Hodgkin lymphoma ,2. In thA surrogate endpoint is a measurement that can be substituted for the final endpoint  to successfully measure the effect of an intervention . Common In 1992, the US Food and Drug Administration (FDA) instituted the accelerated approval process to allow earlier marketing of drugs that treat serious, life-threatening diseases . RecentlWe systematically searched PubMed for articles published between January 1970 and February 2011 to identify experimental or observational prospective studies that assessed OS in MM using TTP, PFS, or EFS as a primary endpoint. Key search words included “myeloma” or “survival” or “progression”; see Additional file Using the median TDE as an independent observation to estimate median OS, with each study arm representing a single observation, presents a statistical challenge. Outcome measures such as TTP, PFS, and EFS differs on their exact definition, especially with the inclusion or exclusion of death as event . ConcernIn addition, the data analyzed are sourced from a literature review of studies with different study designs, patient populations, and treatments. It is possible that the variance of the unexplained share  differs among observations and that heteroskedasticity (non-constant variance) is present. In the presence of heteroskedasticity, the instrumental variables estimator yields consistent, but inefficient, estimates of the coefficients and an inconsistent estimate of the covariance matrix . One wayThe set of available exogenous variables (candidates to instruments) comprise the following: 12-months OS rate; proportion of females; median age; dummy variables characterizing patients by previous treatment; type of TDEs; and publication year.Not all study arms included in the review reached the median OS by the last published follow-up; those that did not are censored observations. If, at the end of a given study, less than 50% of the patients at risk were alive, then the exact value of the dependent variable being modeled (median OS) is known. However, if the duration of the study is shorter than the number of months needed to have less than 50% of patients alive, then all that is known about the dependent variable is that it is higher than the study duration. This information is potentially relevant and was included in our analysis.The estimation of the censored model with one endogenous variable (TDE) was performed by a two-step estimation process similar to the method developed by Smith and Blundell , with th(i) The regression of the endogenous variable on the excluded instruments and exogenous variables (Step 1) was weighted by the number of patients in each study arm and estimated using the GMM Cragg estimator . This es(ii) Given that the censoring point varies with the study arm, a censored normal-weighted regression with the robust option  was estiFollowing the approach described in Smith and Blundell (1986) , variancEvidence in favor of the use of the instrumental variable approach was generated by the Durbin-Wu-Hausman test for endogeneity of the TDE regressor -26. The Alternative model specification was based on three criteria: validity of the instruments used; explanatory power of the instruments in the first-stage regression; and AIC (Akaike Information Criteria) in the second-stage (censored) regression.The association between median TDE and median OS was quantified through Spearman’s rank correlation coefficient in a restricted subset of data consisting of trials only with simultaneously observed values for median TDE and median OS, and excluding those trials with unobserved median OS values ,31. ThisOf the 1,061 studies retrieved, we included 153 studies involving 230 study arms  was highly significant for all TDEs, with a moderate correlation between median OS and median TTP (ρ = 0.51), and a strong correlation for median PFS (ρ = 0.75) and median EFS (ρ = 0.84).The Spearman’s rank correlation coefficient of the aggregated median TDE data on median OS was 0.78 P < 0.0001). Figure . Figure β) correspond to the estimated effect of each variable controlled for the effect of all other variables included in the model. The 95% CIs including zero identify a non-significant effect. We found an increase of 2.45 months  in the reported median OS for each additional month in the observed median TDE (Table Table β were highly significant (P < 0.0001). The inclusion of TTP and EFS as covariables relative to PFS revealed no statistical differences in the type of TDE but a numeric increment of the estimate coefficient of the linear regression relative to the base model. These results suggest that in trials using PFS, the quantitative relationship between PFS and OS is not statistically different to the comparison between TTP or EFS, and OS. However, caution is recommended in interpreting this result because the numeric differences may point to relevant differences in bigger samples. Augmenting the model with information on MM treatment with Thal, Bort, or Len (sample with censored observations) increased the TDE coefficient from 2.36 to 2.45. Despite these therapies not being statistically significant relative to other treatments, they were retained in the model to control for unobserved differences in trials using newer treatment options compared with older treatments. This does not imply that Thal, Bort, and Len have no effect on OS, but instead suggests that these compounds do not add more explanatory power on median OS other than their effect through median TDE.We also tested our modeling technique against a set of alternative specifications and data samples (censored vs uncensored) to assess the robustness of the quantitative relationship between median TDEs and median OS. The departure model identified in Table β = 2.45; 95% CI, 1.71–3.20) relative to the base model .Overall, the consistency of the estimated values for the effect of median TDE on median OS is evident. The maximum variation in the effect of median TDE on median OS is 34%  represent 38.8% and 46.3%, respectively, of the study arms with censored observations, and 23.9% and 55.2%, respectively, of the study arms without censored observations. The results from the regression models including and excluding the two outliers are quite similar, indicating the small effect of these two observations in the analysis.Additional details of the statistical tests performed to assess the validity of the modeling procedures can be found in the Additional file The detailed predicted median OS and associated 95% CIs based on the observed median TDE for each study arm included in our analysis are presented in the Additional file A selection of RCTs retrieved from three recent reviews -35 invesSince 2007, results from 8 phase III trials investigating Thal combinations in patients with newly diagnosed MM not eligible for transplantation have been reported. With the exception of the studies by Ludwig et al.  and MorgIn Figure A recent review by Reece  identifiThere is a sound body of evidence suggesting that TDEs such as PFS, TTP, and EFS are appropriate surrogate endpoints for OS in several types of cancer ,60-71. HOur study is the first to highlight the value of TDEs in predicting OS in MM and to confirm the recommendations of the American Society of Hematology/US FDA Workshop on Clinical Endpoints in both newly diagnosed or relapsed/refractory MM . We focuIn order to assess our model’s ability to predict OS in different settings, we confronted our estimates with data from studies in relapsed/refractory MM led by Dimopoulos et al.  and RichEstimates of median OS using our model suggest an HR for death of 0.34 for Len plus Dex vs Dex alone (median OS 33 vs 11 months), and 0.55 for Bort vs Dex (median OS 15 vs 9 months), assuming event times are exponentially distributed . In thisIt has been argued that OS is not a realistic endpoint in this setting , especiaWe provide a more straightforward way of calculating the expected effect of treatment on median OS (prior to the observation of mature OS data), by estimating an absolute rather than a relative measure for the quantitative relationship between the median TDE and median OS. This regression model recognizes the influence of subsequent therapies because it estimates a mean effect of median TDE on median OS using OS data published in the literature, which is uncorrected for the effect of non-randomized subsequent treatment options. We estimated an average increment of 2.45 months in median OS for each additional month of median TDE. As previously highlighted, these estimates are valuable to assess the expected impact of treatments on median OS, for example in trials of newly diagnosed MM where median OS may not be reached for several years.Information on survival is essential for clinical trial design , accelerOther studies examining the relationship between TDEs and OS have been reported in metastatic colorectal cancer (mCRC) ,62 and iThe following caveats should be considered when interpreting our results. Although it seems reasonable to question the endogeneity of TDEs as an explanatory variable for OS, this issue has not been addressed in the MM literature. The methodology presented here attempts to solve the endogeneity problem, but its applicability depends on the availability of valid instruments.In this analysis, TDEs include three distinct surrogate endpoints; TTP, PFS, and EFS. The estimated relationship between the TDE and OS represents the relationship between an “average” TDE and OS. Although no statistical differences have been found in modeled OS by the type of TDE, the value of the information is limited. Further studies are necessary, particularly to clarify the data from studies using TTP, both because of the competing risk estimation problems  and the Our analysis includes therapies available over a period of 40 years that demonstrated a wide range of efficacy levels. We attempted to control these differences by using publication year as a covariate. In addition, our censored analysis omitted treatment arms with proportionally longer median OS and therefore may not reflect adequately the impact of newer, more effective therapies. Finally, the majority of the studies did not report whether data for OS included patients who were allowed to crossover between treatment arms. Study designs that include automatic treatment crossover can obscure differences in OS, due to the benefit achieved from subsequent treatments .In conclusion, our analysis confirms the potential value of TDEs  in predicting OS in patients with MM. Additional research is welcomed to refine this model or to identify alternative complementary statistical models. Until such models are available and validated, the quantitative relationship presented here may be of value in the design of clinical trials, indirect drug comparisons, and economic assessment of new MM drugs.The authors declare they have no competing interests.JF coordinated the research project, participated in the conception and design of the study, participated in results interpretation and discussion, and drafted the manuscript. FA participated in design of the study, performed the statistical analysis, and participated in results interpretation and discussion. JMA participated in design of the study, coordinated the literature review, and participated in results interpretation and discussion. FJMC participated in design of the study and coordinated the literature review. DF participated in the literature review and in data extraction. ABSP participated in the conception and design of the study and participated in results interpretation and discussion. RR participated in the literature review and in data extraction. JFRR participated in the literature review and in data extraction. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/122/prepubAdditional Results.Click here for file


PDGFD gene might alter the susceptibility to stroke.Platelet-derived growth factor D (PDGF-D) plays an important role in angiogenesis, vessel remodeling, inflammation and repair in response to injury. We hypothesized that genetic variation in We determined the genotypes of a single nucleotide polymorphism (SNP)  in 1484 patients with stroke  and 1528 control subjects from an unrelated Chinese Han population and followed the stroke patients up for a median of 4.5 years.P = 0.05; additive model: OR 1.24, 95% CI 1.01-1.52, P = 0.04) than wild-type genotype. Further analyses showed that -858AA genotype conferred about 2-fold increase in risk of non-hypertensive ICH . After a median follow-up of 4.5 years, -858AA genotype was associated with a reduced risk of ICH recurrence  in non-hypertensive patients.The -858AA genotype showed significantly increased risk of ICH (dominant model: odds ratio [OR] 1.29, 95% confidence interval [CI] 1.00-1.68, The -858AA genotype is probably associated with risk for non-hypertensive ICH. Further studies should be conducted to reveal the role of PDGF-D at various stages of ICH development--beneficial, or deleterious. Stroke is a leading cause of death and disability in China . The numHypertension is the most important cause for spontaneous, non-traumatic ICH both in western nations and in Chinese population ,10. HowePDGFD with the risk of first-ever ICH in a large case-control stroke study in China. We also prospectively followed patients with primary ICH for a median 4.5 years and assessed the relationships of PDGFD variants to the risk of recurrence.Platelet-derived growth factor D (PDGF-D) is the most recently discovered member of the PDGF family and signals mainly through PDGF receptor-β (PDGFR-β) . PDGF-D The study was approved by both the ethics committee of FuWai Hospital and the local ethics committees of collaborative hospitals. All participants reported themselves to be of Han nationality and provided written informed consent.This is a multicenter study for assessment of risk factors of stroke sponsored by the Ministry of Science and Technology of China (973 Project). Initially, 2000 patients with stroke and 2000 control subjects were recruited as described previously ,16.Stroke was defined as a sudden onset of nonconvulsive and focal neurological deficit persisting for > 24 hours. Cerebral thrombosis was diagnosed if the patient had the characteristics of cerebral cortical dysfunction, and CT scan or MRI performed within 28 days of onset either showed an area of low attenuation in a region compatible with the clinical symptoms or failed to identify an alternative cause of the symptoms. For CT/MRI readers, the intrareader variability was not calculated. Among all subtypes of hemorrhagic stroke, only spontaneous, non-traumatic, primary ICH was in the range of selection. Other types of stroke, including transient ischemic attack, subarachnoid hemorrhage, embolic brain infarction, brain tumors, and cerebrovascular malformation, and severe systemic diseases such as collagenosis, endocrine and metabolic disease (except diabetes mellitus [DM]), inflammation, liver, neoplastic, or renal diseases were in the range of exclusion.Before data analysis, 516 stroke cases and 472 controls were excluded for indefinite diagnosis (24 cases), absence of plasma (76 cases and 93 controls), insufficient DNA (355 cases and 309 controls), and failure of genotyping (61cases and 70controls). No significant differences were found in clinical characteristics between the included and excluded subjects. Finally, 1484 patients  and 1528 controls were eligible for genetic analysis.To assess whether genetic variations contributed to the risk of ICH recurrence after the first-ever ICH, we prospectively followed this stroke population through May 31, 2006 by a standard questionnaire and telephone contact by physician investigators. During a median 4.5 years  of follow-up for 411 patients with ICH, 14 participants were lost and excluded from analysis. Follow-up data were completed for 397 stroke patients .Blood samples were obtained after at least a 12-hour overnight fasting period and pre-chilled vacutainers and immediately centrifuged. The biochemical variable determination and clinical data collection were performed as described previously ,16.PDGF-D gene is located on chromosome 11q22.3 [2 of 0.8 was chosen as a threshold for all analyses. Only one Tagger SNP  with a minor allele frequency more than 5% was genotyped within the promoter region of PDGFD [DNA was extracted from the buffy coat fraction of centrifuged blood using the QIAmp Blood Kit . The  11q22.3 . The selof PDGFD . Sequenc[Additional file 1].Detailed descriptions of genotyping were shown in PDGFD promoter (from -1168 to +185) in HCT-116 cells. The detailed descriptions of the luciferase reporter assays were shown in [Additional file 2].We investigated whether the variant at position -858 affects the transcriptional activity of the t test or one-way ANOVA, and the categorical variables were compared by chi-square test.The distribution of continuous variables was tested for normality with a one-sample Kolmogorov-Smirnov test. Because the level of triglyceride was highly skewed, the Mann-Whitney nonparametric method was applied to compare the differences between patients and controls. Continuous variables were compared by unpaired Student Hardy-Weinberg equilibrium of each variant was tested using chi-square test. Odds ratios (ORs) and 95% confidence interval (CI) were used to determine the association between genotypes and the risk of ICH. Multivariate unconditional logistic regression was used to estimate OR and 95% CI after adjustment for age, gender, body mass index (BMI), hypertension, DM, HDL cholesterol (HDL-C), Non-HDL cholesterol (non-HDL-C), total cholesterol (TC), triglyceride (TG), drinking and smoking status. Multiple testing was adjusted using Bonferroni correction.The Kaplan-Meier analysis and Cox proportional hazards models were used to describe the association of genotypes with incidence of ICH recurrence and cardiovascular deaths. Follow-up began on the date of diagnosis of the first index ICH and continued until the date of a recurrent stroke or the end of follow-up period. Hazard Ratio (HRs) and 95% CI were estimated using multivariate Cox proportional hazards models after adjustment for age, gender, BMI, hypertension, HDL-C, TC, TG, DM, smoking status, and alcohol intake.P < 0.05 is considered statistically significant. Analyses were performed with SPSS software version 11.0 , and Stata software version 9.0 . The power analysis was performed with the software Quanto (http://hydra.usc.edu/gxe/).All reported P values are 2-sided, and [Additional file 3]. The demographic and clinical characteristics of the subjects in the present study are shown in Table Proportions from each of the clinical sites for cases and controls were shown in The present study had > 80% power to detect an association with ORs of ≥ 1.2 for alleles at 10% to 40% frequency for the total population and the subgroup analysis.PDGFD genotypes of -858 is shown in Table vs. AC vs. AA) and recessive model (CC+AC vs. AA). The additive genetic model assumes that there is a linear gradient in risk between the CC, AC and AA genotypes (CC genotype baseline). After adjustment for age, gender, BMI, smoking, HDL, non-HDL-C, TC, TG, blood pressure and fasting blood glucose with multivariate logistic regression analysis, the presence of the AA genotype of the -858 locus showed significantly increased risk of ICH  than wild-type genotype.The distribution of P = 0.001; additive model: OR 1.75, 95% CI 1.24-2.46, P = 0.001; recessive model: OR 1.77, 95%CI 0.79-3.95, P = 0.17)  ICH and 97 were non-hypertensive. Further analyses showed that -858AA genotype conferred about 2-fold increase in risk of non-hypertensive ICH  of follow-up for 397 patients, a recurrent ICH occurred in 52 persons , of which 39 were baseline hypertensive cases and 13 were baseline non-hypertensive. The risk time for recurrence in hypertensive and non-hypertensive ICH were shown in more detail PDGFD -858 variant with ICH recurrence was found in hypertensive patients.Among patients with non-hypertensive ICH, the variant -858AA genotype was significantly associated with reduced risk of ICH recurrence Figure , which wPDGFD promoter in HCT-116 cells. The variant-858A-bearing PDGFD promoter exhibited a 3.78-fold increase in transcription activity than the variant-858C-bearing promoter. The pGL-3 empty vector had a negligible amount of the luciferase activity (Figure We investigated whether the polymorphism at position -858 affects the transcriptional activity of the y Figure . The resPDGFD gene was found to be associated with more than 2-fold increase in risk of first-ever ICH in non-hypertensive cases, while reduced incidence of recurrent ICH.The present study is the first clinical investigation of the significance of PDGF-D regarding the incidence and prognosis of ICH in a Chinese population. A common functional variation in the promoter region of PDGFD promoter region could lead to an enhancement of PDGFD promoter activity and consequently up-regulation of the expression of the gene. Interestingly, this functional variant was only associated with non-hypertensive cases, indicating that there are significant differences in genetic background, mechanisms and pathologies between the hypertensive and non-hypertensive ICH.In a small sample of south China, Han et al. have found that the G allele of rs7950273 of the PDGFD is associated with higher risk of ischemic stroke. However, the associations of PDGFD variants with other type of stroke, especially ICH have not been investigated . In the In this study, the relations between PDGFD variation and recurrent ICH were quite contrary to the contribution of the variant to the risk of first-ever ICH, which can probably be explained by the angiogenic and wound healing effects of PDGF-D. PDGF-D induces macrophage recruitment, extracellular matrix deposition, and blood vessel maturation, which are all part of process of angiogenesis and wound healing ,19. In aTherefore, one can speculate that the enhancement of PDGF-D function is associated with proliferation and migration of SMCs, deposition of extracellular matrix, and degradation of vascular basement membrane, which on one hand lead to a loss of vessel wall integrity, resulting in increased susceptibility to primary non-hypertensive ICH. On the other hand, the above processes are also important procedures for angiogenesis and are essential for the development of neovessels during the restoration of vasculature system of injured brain after primary ICH ,22.The present study has limitations. First, although the sample size of this study is large, the relatively small number of each stroke subtype, especially the small sample size of non-hypertensive ICH, is a limitation of the present study; therefore further large scale studies focused on non-hypertensive ICH will be needed. Second, multiple testing of the genetic associations must be noted. After Bonferroni correction for multiple testing, the only association that maintains is the association with non-hypertensive ICH. Third, for CT/MRI readers, the intrareader variability was not calculated. However, the confirmed diagnosis of each case was based on results of at least two CT/MRI readers and one neurologist; disparities were resolved by discussion. The accuracy of the diagnosis was guaranteed by these methods.PDGFD -858A/C variant is probably associated with risk for non-hypertensive ICH. Further studies should be conducted to reveal the role of PDGF-D at various stages of ICH development--beneficial, or deleterious.In conclusion, our observations suggest that The authors declare that they have no competing interests.BY carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. CJ participated in the sequence alignment and was involved in critically revising the manuscript for importand intellectual content. SK and WY participated in the design of the study and performed the statistical analysis. HR conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.Genotyping of -858 Site. Methods of genotyping and sequencing analysis of the -858 site of PDGFD.Click here for fileLuciferase reporter assays. The detailed descriptions of the luciferase reporter assays.Click here for fileProportions from each of the clinical sites. The detailed descriptions of the proportions from each of the clinical sites for cases and controls.Click here for fileThe risk time for recurrence in ICH. The detailed descriptions of the risk time for recurrence in hypertensive and non-hypertensive ICH.Click here for file


Background: Changes in fiber tract architecture have gained attention as a potentially important aspect of schizophrenia neuropathology. Although the exact pathogenesis of these abnormalities yet remains to be elucidated, a genetic component is highly likely. Neuregulin-1 (NRG1) is one of the best-validated schizophrenia susceptibility genes. We here report the impact of the Neuregulin-1 rs35753505 variant on white matter structure in healthy young individuals with no family history of psychosis. Methods: We compared fractional anisotropy in 54 subjects that were either homozygous for the risk C allele carriers (n = 31) for rs35753505 or homozygous for the T allele (n = 23) using diffusion tensor imaging with 3T. Tract-Based Spatial Statistics (TBSS), a method especially developed for diffusion data analysis, was used to improve white matter registration and to focus the statistical analysis to major fiber tracts. Results: Statistical analysis showed that homozygous risk C allele carriers featured elevated fractional anisotropy (FA) in the right perihippocampal region and the white matter proximate to the left area 4p as well as the right hemisphere of the cerebellum. We found three clusters of reduced FA values in homozygous C allele carriers: in the left superior parietal region, the right prefrontal white matter and in the deep white matter of the left frontal lobe. Conclusion: Our results highlight the importance of Neuregulin-1 for structural connectivity of the right medial temporal lobe. This finding is in line with well known neuropathological findings in this region in patients with schizophrenia. The schizophrenias are a group of—most likely pathophysiologically and etiologically heterogeneous—disorders that go along with progressively disabling deficits in cognition and behavior. Although the exact etiopathogenesis yet remains to be fully elucidated, current literature suggests the view of largely genetically determined disorders with changes in brain structure and function   gene is an interesting candidate in this context. It is assumed that mutations in the The original core haplotype consisted of five single-nucleotide polymorphisms (SNPs) and two microsatellite markers. Of all studied markers of the NRG1 genomic region, rs35753505, which is located in the 5′-flanking region of NRG1, is the most commonly reported single marker. Even though some authors found strong associations of rs35753505 with schizophrenia, others failed to do so  allele carriers for this polymorphisms, while not considering heterozygous allele carriers in this study.Readdressing the heterogeneous results of previous studies on NRG1 rs35753505 variant. The subjects had a mean age of 22.9 years (SD = 2.8), were right handed , and had 15.6 (2.3) years of education. Their fathers were educated for 16.0 (4.4) and their mothers for 14.4 (4.3) years on average. Mean intelligence quotient (IQ) was 112.1 (12.2). After a complete description of the procedure, subjects provided written informed consent to participate in the study  underwent DTI after genotyping for the GCCAAAT-3′; 5′-FAM-CATGTATCTTTATTTTACCAAAT-3′. Sequence information was obtained from the homepage of deCODE Genetics (http://decode.com/nrg1/markers/SNPS.htm). Replication of 15% of the sample showed no differences in genotypes.The rs35753505 was genotyped using Applied Biosystems 7900HT Fast Real-Time polymerase chain reaction (PCR) System and TaqMan-probes designed by Applied Biosystems . Primers and VIC/FAM-probe sequences for rs35753505 detection were as follows: Forward-5′-TTTAAGGCATCAGTTTTCAATAGCTTTTTTATGT-3′; Reverse-5′-AGACAGATGTCTCAAGAGACTGGAA-3′;5′-VIC-CATGTATCTTTATTTTb-value of 800 sec/mm2, in addition seven interleaved acquisitions of non-DW images (b = 0). The protocol was acquired four times and, after individual motion correction, the DW images were averaged to increase the signal-to-noise-ratio. In addition, an anatomical T1-weighted magnetization prepared rapid gradient echo sequence was acquired (1 mm isotropic resolution).Imaging was performed on a 3-Tesla Trio MR scanner  in the Institute of Neuroscience and Biophysics—Medicine, Research Center Jülich. Subjects for the scans were chosen due to their genotype. Head movements were minimized by immobilizing the head during the scanning procedure using foam cushions. Images were acquired with a diffusion-weighted (DW) double spin-echo echo planar imaging sequence . A 12-channel phased-array coil was used and the sequence utilized twofold acceleration with the GRAPPA parallel imaging technique  on the individual FA maps -style DTI analysis —option as implemented in FSL. To control for gender effects, subjects' sex was included as a covariate of no interest into the statistical model. The t-maps were then thresholded at P (uncorr.) <0.001 and projected onto the mean FA skeleton for visualization.Comparison between the homozygotic wild type T allele carriers and homozygotic risk C allele carriers was performed by means of a two-sample non-parametric For a more detailed anatomical analysis, we used the Anatomy Toolbox  or gender (P = 0.503). Also mean IQ  was not significantly different in both groups (P = 0.8).The images of 54 subjects  were included in our TBSS analysis. The homozygous wild-type group consisted of 31 subjects , the homozygous risk allele carrier group consisted of 23 subjects . Mean age in the wild-type group was 23.1 years (SD: 3.2 years), in the homozygous risk allele carrier group 22.6 (SD: 2.2 years). Both groups did not differ significantly in mean age (k = 504), while one cluster was situated in the white matter proximate to the left area 4p . The smallest cluster was located in the right hemisphere of the cerebellum  . Another cluster was located in the right prefrontal white matter . A third cluster was situated in the deep white matter of the left frontal lobe   rs1018381 schizophrenia susceptibility variant, which was associated with FA reductions in the right perihippocampal region .There are several limitations in this study. First, the changes in brain structure we found were rather discrete. It has to be considered however that we examined healthy young subjects with a high level of intellectual functioning and without history of psychiatric disorder in first degree relatives.Moreover, it needs to be pointed out that our results were not corrected for multiple comparisons. However, the significance threshold was comparable to the study of Winterer et al.  and evenThe fact that these results were not so pronounced as to survive correction for multiple comparisons raises the problem of false positive findings. Underpowered studies due to small sample sizes can be a critical factor in the generation of false positive results. This becomes even more problematic when the effects studied are rather subtle. Given the rather low odds ratios of many schizophrenia susceptibility gene variants, also sample sizes that are usually regarded as sufficient in structural imaging studies can thus be relatively small and entail a potential danger false positive findings. The balance between controlling type I and type II errors is indeed a pertinent problem in neuroimaging. Much of this is related to the fact that, in particular at currently employed finer spatial resolution, the number of assessed voxels and hence the number of parallel tests are extremely high . This renders correction for multiple comparisons very conservative and biased toward false negative findings. It also needs to be pointed out that due to the indirect nature of the diffusion MR signal as a proxy measure for fiber tract integrity and in particular the usually “relatively” low sample size (including random effects from sampling) limit the capacity to completely exclude false positive findings even despite conservative thresholding. Conversely, more liberal thresholds obviously entail the increased danger of identifying random noise in the data, for example, due to the sampling of the subjects, as true effects. Importantly, however, such effects should not be reproducible across studies. In other words, even highly conservative inference, bringing with it a high danger of false negatives, may not necessarily protect against effects due to random sampling of a relatively small group from the underlying population. Importantly, these effects would not be false positives in the statistical sense , but still would reflect findings that are not reproducible in further studies from the underlying general population. One potential way to overcome this predicament not only in diffusion analysis but also in neuroimaging per se is the focus on consistency of findings across studies (Eickhoff et al. Although our sample consisted of populations that could potentially differ in the frequency of the risk allele due to a different ethnical background, we did not genotype the sample for ancestry informative markers. Consequently, effects of ancestry could have led to additional variance in our data.We included both male and female subjects in this study. Although gender was used as a covariate, a direct comparison between male and female T allele carriers would be interesting. However, the number of subjects included does not allow such a comparison. Future studies enrolling larger populations should also focus on gender-specific effects.In summary, we found both clusters of elevated and reduced FA in NRG1 rs35753505 C allele risk type carriers. Changes were most pronounced in the right perihippocampal region, where risk type carriers showed elevated FA values. The structural alterations described might in part be responsible for differences in BOLD response found by functional imaging studies in a largely overlapping population (Krug et al.