Elevated high-sensitivity C-reactive protein (hsCRP) increases the risk of cardiovascular disease (CVD) in the general population, but its role as a predictive marker in HIV-positive patients remains unclear. Aim of the study was to evaluate whether hsCRP or other biomarkers are independent predictors of CVD risk in HIV-infected patients.Retrospective, nested case–control study. HIV-positive men and women (35–69 years of age) receiving combination antiretroviral therapy (cART) were included. Cases (n = 35) had a major CVD event. Controls (n = 74) free from CVD events for at least 5 years from starting ART were matched on diabetes and smoking. HsCRP, D-dimer, P-selectin, interleukin-6 (IL-6), tissue plasminogen activator, plasminogen activator inhibitor-1 levels were measured.P = 0.03). Higher IL-6 and P-selectin levels were also independently associated with increased CVD risk, although the association was weaker than for hsCRP. Higher total cholesterol and lower HDL cholesterol increased CVD risk, independent of hsCRP.High hsCRP was associated with CVD risk, independently of traditional cardiovascular risk factors, HIV replication and the type of ART received at the time of sampling (adjusted odds ratio 8.00 [1.23-51.94] comparing >3.3 mg/L with <0.9 mg/L; hsCRP may be a useful additional biomarker to predict CVD risk in HIV-infected patients receiving cART. Combination antiretroviral therapy (cART) has substantially reduced HIV-related morbidity and mortality . As a reAlthough several studies have suggested a positive association between cART and CVD risk ,11,12, tResearch into CVD risk factors in the general population has identified a number of predictive biomarkers, such as apolipoprotein B, total cholesterol, interleukin-6 (IL-6), serum amyloid A, D-dimer, tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), P-selectin, intercellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1) and, most importantly, C-reactive protein (CRP) as determined by high sensitivity techniques (hsCRP) -29. HsCRGiven the increased risk of CVD in HIV-infected patients, the predictive role of such biomarkers in this patient population is of interest. Several potential CVD markers have been investigated in HIV-positive patients, including lipoprotein particles, measurements of carotid artery intima–media thickness and of arterial stiffness, tumor necrosis factor-α, IL-6, IL-10, myeloperoxidase, platelet (P)-selectin, leptin, D-dimer, adiponectin, soluble VCAM-1, ICAM-1 and ICAM-3, and hsCRP -33. A reOur primary objective was to evaluate whether hsCRP predicts CVD risk in HIV-infected patients, independently of other established risk factors, using the data of persons enrolled in two Italian cohorts. Our secondary objective was to investigate the association between other biomarkers and CVD.We studied patients enrolled in two cohort studies; the Icona Foundation Cohort and the Catholic University of the Sacred Heart (CUSH) clinical database, Rome. In CUSH, data have been collected as part of routine clinical care since 1997 (full details are reported elsewhere ). The IcAll patients provided written consent to participate in Icona Foundation Study and in CUSH, following procedures in accordance with the ethical standards of the responsible committee on human experimentation and the Helsinki Declaration. In both studies, patients informed consent includes a specification for use of samples for further research in HIV. No specific consent for inclusion in the current analysis was therefore needed. The studies and the corresponding informed consents were approved by the Ethics Committees of the various ICONA clinical sites (see Appendix) and that of the Catholic University for CUSH. Inclusion criteria for this analysis were age 35–69 years and no history of a major CVD event before starting cART and no evidence of any other inflammatory disorder over the 3 months prior to the date of the stored plasma sample. Exclusion criteria were current use of active hormonal-based therapy and of therapy with anti-inflammatory drugs or statins. A small proportion of patients had used these drugs prior to the date of the stored sanple.We conducted a retrospective, matched case–control study nested within CUSH and the Icona Foundation Study involving HIV-infected patients with CVD (cases) or without CVD (controls). CVD cases were defined as HIV-infected patients who had a major cardiovascular event  while receiving cART , and for whom at least one plasma sample was stored before the CVD event. Within each cohort, HIV-infected controls (a minimum of two per each case) were defined as patients who were currently free of CVD events (i.e. had never developed CVD events), had at least 5 years of follow-up after cART initiation and had at least one plasma sample stored over the observation period.Cases and controls were matched on previous history of diabetes and smoking status. The ‘index time’ for analysis was fixed at the date of the CVD event for cases and at last follow-up for controlsBecause date of sample for controls was on average much more recent than that of cases, calendar year and time between the date of sample and the index time were included as an additional adjusting factor in the multivariable analyses.In CUSH, plasma samples are collected each time a viral load assay is performed (i.e. approximately every 3 months), and stored at −80°C. In the Icona Foundation Study, plasma samples are stored at the time of enrolment and at least once a year. In both cohorts, plasma samples are collected using Vacutainer® collection tubes . In the case of ICONA foundation Study, samples are initially kept frozen at the various clinical sites at −20°C or −80°C and subsequently shipped using dry ice to a central repository in Rome, Italy, where they are kept at −80°C until use for retrospective analysis. Dates of sampling were (ranges) 1999–2008 for cases and 2006–2008 for controls for CUSH, and 1998–2004 for cases and 2000–2004 for controls for the Icona Foundation Study. As a general rule, the most recent plasma sample stored before the analysis index time was analyzed in both cases and matching pair of controls (late samples). However, for some cases and controls, if more than one stored sample was available, biomarkers were measured using an additional earlier sample (early samples). To avoid bias, laboratory personnel was blind to whether the sample was coming from a case or a control. Samples, which were labelled with a unique identifier so that they could be linked with patients ID and their clinical data for statistical analysis, were analyzed in random order.Total cholesterol and HDL were measured using standard techniques by local laboratories, while biomarkers were analyzed by the Laboratory of Genetic and Environmental Epidemiology, Catholic University of Campobasso, Italy. hsCRP was measured by nephelometry using an automatic analyzer . Quality control was performed using an internal laboratory standard; the inter-day coefficient of variation (CV) was 2%. D-Dimer was evaluated utilizing a latex particle-enhanced immunoturbidimetric assay . Quality control was performed using an in-house citrate plasma pool; inter-and intra-day CV was 7.6% and 5.4%, respectively. P-selectin , IL-6 , t-PA and PAI-1  were measured utilizing commercially available enzyme-linked immunosorbent assay (ELISA) kits, as specified by the manufacturers. The intra-assay CV, using in-house citrate plasma, were, according to the manufacturer, 4.3%, 10.5%, 4.8% and 9.8% respectively.The detection limit of the different assays were 1.06 ng/ml for P-selectin, 1.0 ng/ml for t-PA, 1.0 ng/ml for PAI-1 and 0.70 pg/ml for IL-6.By setting the type I error α at 5%, and assuming that 40% of patients had a hsCRP level above 3 mg/L  and two Summary statistics and illustrations appropriate for matched case–control studies were used to summarize key findings . Conditi2 transformed biomarker levels were also used, estimating the increased risk of CVD associated with a one log2 difference in biomarker levels. Separate models were fit for each biomarker and, because of the large number of parameters involved, we decided a priori not to fit the model mutually controlling for all biomarkers. The following covariates assessed at the date of stored samples were considered for the adjusted analyses: calendar year, duration of time between the date of sample and the analysis time, age, total cholesterol, HDL, CD4+ cell count and viral load, cumulative exposure to nonnucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitor (NRTIs) and protease inhibitor (PIs) prior to sample, and co-infection with hepatitis B or C. [clogit in STATA [Models with log B or C. . Sensitiin STATA . All staWe studied 109 patients  of whom 17 cases, 40 controls were from CUSH and 18 cases, 34 controls from the Icona Foundation Study. The distribution of the matching variables in cases and controls was: smokers/diabetics , non-smokers/diabetics , smokers/non-diabetics , non-smokers/non-diabetics . Characteristics of cases and controls at the time of the late samples are summarized in Table P = 0.09) and late samples . Higher median values were observed for t-PA in early samples  and P-selectin in late samples  while the 74 controls contributed 145 samples (74 early and 71 late samples) for biomarkers analysis. In the unadjusted analysis comparing median values of biomarkers between cases and controls, the only biomarker consistently showing a higher median value in cases compared with controls was hsCRP, both in the analysis using the early samples  had levels in the lowest tertile compared with 26/74 (35% of controls). In contrast, the corresponding percentages with levels of hsCRP falling in the highest tertile were 14/35 (40%) and 26/74 (26%) for cases and controls, respectively. Similarly, for the late samples alone, 5/33 (15%) of cases and 23/71 (32%) of controls had hsCRP values in the lowest tertile vs. 14/33 (42%) and 14/71 (18%) in the highest tertile. For the combined data set, the corresponding values were 11/68 (16%) for cases and 49/145 (34%) for controls with hsCRP levels in the lowest tertile vs. 28/68 (41%) and 33/145 (23%) in the highest tertile.Based on univariable and multivariable analyses, on late samples Table  and the None of the other considered biomarkers showed an independent association with the risk of CVD. Covariate adjustment showed that the magnitude of the association was underestimated in the unadjusted analysis as adjusted OR were even higher, especially for hsCRP. Use of the combined data set provided more precise estimates, as a result of increased statistical power Tables a and b.P = 0.112) for values of 1–3 mg/L and 13.69  for values >3 mg/L, as compared to values of <1 mg/L. The corresponding values for the combined data set were 6.5  and 11.4 .Sensitivity analyses using the clinically validated hsCRP categories  showed s2 higher hsCRP, adjusted OR 1.45 , p=0.032), for eGFR  and for prior use of statins .We performed additional sensitivity analyses on the combined early and late samples dataset. In the multivariable models, a similar association of hsCRP levels with the risk of CVD events was observed after further adjusting for BMI (per logP = 0.001), a lower HDL cholesterol , a less recent calendar year of the stored sample  and shorter time between the date of sample and the analysis time .In the main multivariable model including hsCRP as a continuous variable , and using the combined data set Table b, the foFrom the analysis using the combined set of early and late samples, we estimated that elevated hsCRP was associated with increased CVD risk in HIV-positive patients receiving cART (8-fold risk increase comparing patients with hsCRP levels >3.3 mg/L with those with <0.9 mg/L). A key finding was that the effect of hsCRP on CVD risk appeared to be independent of traditional CVD risk factors , as well as potential confounders, such as BMI, renal function and use of statins. Increased risk of a CVD event was also evident for higher values of IL-6 and P-selectin, although the association was weaker than for hsCRP. In addition, higher total cholesterol and lower HDL cholesterol were confirmed to be independent predictors of risk of CVD in our HIV-positive population.The collection of early and late samples provided an opportunity to investigate whether the prognostic value of the studied biomarkers may differ according to specific objectives (i.e. to predict the short-term vs. the long-term risk of CVD). In the unadjusted analysis, median hsCRP levels were higher in cases (i.e. with CVD events) than controls (i.e. without CVD events) for both early and late samples, and more cases had hsCRP levels in the highest vs. lowest tertile, although this difference was more significant in late samples. Numerical problems due to sparse data in the analysis including only early samples, did not allow the calculation of adjusted estimates in this subset. Despite this limitation, our findings suggest that in HIV-infected patients hsCRP may be a useful marker to predict the medium-to-long term risk of experiencing CVD events , although the crude association of this marker with CVD events was actually stronger for the short term.Several studies have investigated hsCRP levels in HIV-positive patients, and the association between hsCRP and CVD risk. HsCRP levels were not statistically different between untreated HIV-infected and uninfected individuals in one study , while ilow risk of a CVD event in the study by Boger et al. [In the analysis by Triant et al. including HIV-positive and -negative patients enrolled in a large US healthcare system, elevated CRP levels were associated with more than a four-fold increase in the risk of acute myocardial infarction compared with HIV-negative patients with normal CRP , but ther et al. .Similar to hsCRP, IL-6 is a marker of inflammation and has been associated with coronary heart disease and endothelial dysfunction : therefoThe limitations of the current study include the inability to assess the risk of CVD associated with gender  and the matching variables (smoking status and diabetes). Also, we were unable to control our estimates for potential differences in systolic blood pressure because a measure of this parameter was available only in a minority of the patients studied (not shown). Furthermore, time from baseline was not one of the matching criteria as it is generally the case in case–control studies nested within a prospective cohort, although cases and controls were matched within the same cohort. In addition, controls have been, on average, exposed to cART for longer than cases , and, as a consequence, biomarkers have been measured in more recent calendar years. However, both factors were controlled for in the multivariable analysis. Another limitation relates to the different number of samples contributed by patients to the analysis, although there were only minimal imbalances. Finally, the fact that no association was found between t-PA, PAI-1 or D-dimer and CVD risk in this study does not exclude their role, as the study was not powered to detect such associations.The importance of evaluating, preventing and managing CVD in patients with HIV is recognized in guidelines for the use of antiretroviral agents in HIV-infected adults . BecauseIn summary, our analysis shows that hsCRP is a candidate biomarker predicting CVD risk in HIV-infected patients receiving ART. Additional studies analyzing the influence of the addition of this marker on the prediction of cardiovascular risk scores are required before implementation of routine measuring for the scope of prediction into clinical practice.Members of the ICONA Foundation StudyGoverning bodyM. Moroni (Chair), G Angarano, A Antinori, F Castelli, R Cauda, A d’Arminio Monforte, G Di Perri, M Galli, R Iardino, G Ippolito, A Lazzarin, CF Perno, O Armignacco, PL Viale, F Von Schlosser.Scientific secretaryA d’Arminio MonforteSteering committeeA Ammassari, M Andreoni, A Antinori, C Balotta, P Bonfanti, S Bonora, M Borderi, MR Capobianchi, A Castagna, F Ceccherini-Silberstein, P Cinque, A Cozzi-Lepri, A d’Arminio Monforte, A De Luca, M Gargiulo, C Gervasoni, E Girardi, A Gori, G Guaraldi, M Lichtner, S Lo Caputo, G Madeddu, F Maggiolo, G Marchetti, S Marcotullio, L Monno, R Murri, C Mussini, M Puoti, C TortiStatistical and monitoring teamA Cozzi-Lepri, P Cicconi, I Fanti, T Formenti, L Galli, P LorenziniParticipating physicians and centersItaly A. Giacometti, A Costantini, A. Riva (Ancona); G. Angarano, L Monno, C Carrisa, (Bari); F. Maggiolo, G Lazzari (Bergamo); PL. Viale, M Borderi, G. Verucchi (Bologna); F Castelli, C. Torti, C. Minardi, (Brescia); T. Quirino, C Abeli (Busto Arsizio); P.E. Manconi, P. Piano (Cagliari); J Vecchiet, K Falasca (Chieti); L. Sighinolfi, D. Segala (Ferrara); F. Mazzotta, S. Lo Caputo (Firenze); G. Cassola, G Viscoli, A. Alessandrini, R. Piscopo, G Mazzarello (Genova); C. Mastroianni, V. Belvisi (Latina); P. Bonfanti, I. Caramma (Lecco); A. Chiodera, P. Castelli (Macerata); M Galli, A. Lazzarin, G. Rizzardini, M. Puoti, A. d’Arminio Monforte, AL Ridolfo, R Piolini, A Castagna, S Salpietro, A Galli, A Bigoloni, V Spagnuolo, L Carenzi, P Zucchi, M.C. Moioli, R Rossotti, P Cicconi, T Formenti (Milano); C. Mussini, L Bisio (Modena); A Gori, G Lapadula (Monza), N. Abrescia, A. Chirianni, MG Guida, M Gargiulo (Napoli); F Baldelli, B Belfiori (Perugia); G. Parruti, T Ursini (Pescara); G. Magnani, M.A. Ursitti (Reggio Emilia); R. Cauda, M Andreoni, A. Antinori, V Tozzi, V. Vullo, A. De Luca, A. d’Avino, M. Zaccarelli, L Gallo, E. Nicastro, R. Acinapura, M Capozzi, R Libertone, M. Lichtner, G Tebano, (Roma); M.S. Mura, G Madeddu (Sassari); P. Caramello, G. Di Perri, G.C. Orofino, M Sciandra (Torino); G. Pellizzer, V. Manfrin (Vicenza).Ethics Committees of the Clinical Centers participating to the ICONA Foundation StudySpedali Civili - P.zza Spedali Civili, 1 25125 – BresciaOspedale Amedeo di Savoia - C.so Svizzera, 164–10149 TorinoOspedale A Manzoni - Via dell’Eremo 9/11 - 22053 LeccoOspedale Busto Arsizio - Piazzale Solaro, 3–21052 Busto Arsizio (VA)Policlinico Sant’Orsola Università di Bologna - Via Massarenti, 11–40138 BolognaArcispedale S Maria Nuova - V.le Risorgimento, 80 42100 Reggio EmiliaAzienda Policlinico di Modena -Via del Pozzo, 71–41100 – ModenaAzienda Ospedaliero Universitaria di Ferrara Via Aldo Moro, 8 44124 Cona - FerraraUniversità Politecnica Marche - Ospedale Umberto I L. go Cappelli, 1–60121 AnconaAzienda Ospedaliera Universitaria Osp Riuniti di Ancona - 60020 - Torrette di AnconaPresidio Ospedaliero AUSL 9 - Via Santa Lucia - 62100 MacerataIstituto Nazionale per le Malattie Infettive IRCCS “L Spallanzani ”-Via Portuense, 292–00149 RomaUniversità di Perugia Policlinico Monteluce - Via A. Brunamonti - 06122 PerugiaPoliclinico Umberto I, Univ La Sapienza - V.le del Policlinico, 155–00163 RomaFondazione PTV - Policlinico Tor Vergata - Viale Oxford, 81–00133 RomaUniversità “Aldo Moro” - P. le G. Cesare, 11–70124 BariAzienda Ospedaliera Cotugno - Via G. Quagliarello - 80131 NapoliUniversità “G D’Annunzio” - Via Valignani - 66100 ChietiUSL di Pescara VIA FONTE ROMANA, N ° 8–65124 PESCARAth, 2010 to Feb 28th 2011. ADM received speaker honoraria and fees for attending advisory boards from GlaxoSmithKline, Gilead, Bristol-Myers-Squibb, Boehringer-Ingelheim, Abbott Virology, Tibotec.ADL received speaker honoraria and fees for attending advisory boards from ViiV Healthcare, Gilead, Abbott Virology, Janssen and Siemens Diagnostics. MC has been a paid consultant for Merck Sharp & Dohme, Italy and has been employed by Bristol-Myers-Squibb, Italy since May 10No other member of the Icona Foundation Study has any financial or personal relationships with people or organizations that could inappropriately influence this work, although most members of the group have, at some stage in the past, received funding from a variety of pharmaceutical companies for research, travel grants, speaking engagements or consultancy fees.Conception and design: ADL, KGD, MC, Ad’AM. Analysis and interpretation of the data: AC-L, ADL, LI, ADC, Ad’AM. Critical review and revision of the article for scientific accuracy and intellectual content: all. Final approval of the article: all. Statistical expertise: AC-L. Administrative, technical or logistic support: MC, RC, Ad’AM. Collection and assembly of data: ADC, MC, KGD, MP, AC-L, Ad’AM, AG, FG, GS.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/414/prepub


The 32Q  and 32W  variants of complement factor B (CFB) cause less efficient complement activation in vitro than the common 32R variant. This is thought to be the reason that the 32Q variant is associated with decreased risk of age-related macular degeneration (AMD). We investigated whether the 32W variant was also associated with decreased risk of AMD.CFB alone and in combination with smoking status and genetic risk markers of complement factor H (CFH) and HtrA serine peptidase 1 (HTRA1). We performed meta-analysis of all previously published reports of 32W allele frequency in AMD cases and controls.We genotyped 367 cases with neovascular AMD and 251 disease-free controls. Association with the disease phenotype was assessed by logistic regression for polymorphisms of CFH haplotypes, HTRA1 rs10490924 genotype, and smoking status). Meta-analysis  including this study and two others of neovascular AMD showed a combined odds ratio of 0.75 (p<0.05) for 32W, compared to 32R. Meta-analysis  of all reported studies of all types of AMD showed a combined odds ratio of 0.79 (p<0.01).The CFB variant 32W was associated with protection against neovascular AMD, compared to the common 32R variant (odds ratio 0.64, p<0.05, in logistic regression with CFB variants; odds ratio 0.53, p<0.05, in logistic regression with CFB variants, Our study shows that the 32W variant of CFB is associated with protection against AMD, in keeping with evidence of its functional effect on the complement system. The protective effect is less strong than that associated with 32Q. CFH) gene and susceptibility to the disease [CFI) [CFB) [C3) [CFHR3), and complement factor H-related 1 (CFHR1) [CFH, the deletion of CFHR3 and CFHR1 [CFB [Age-related macular degeneration (AMD) is a major cause of visual impairment and blindness among older people . The alt disease -8. Sincese [CFI) , complemI) [CFB) , complemFB) [C3) , complem (CFHR1)  genes ha (CFHR1) -15 polymnd CFHR1 , and theHR1 [CFB .rs641153 (R32Q) and rs12614 (R32W), affect the same codon and amino acid residue. Of the four possible combinations of bases, three are found to exist, with the combination of both major alleles resulting in an arginine (R), the minor allele of rs641153 with the major allele of rs12614 resulting in a glutamine (Q), and the minor allele of rs12614 with the major allele of rs641153 resulting in a tryptophan (W) at residue 32  in AMD.The central step of all three complement pathways is activation of the C3 molecule to C3b by a C3 convertase enzyme  studied sidue 32 . This reThe characteristics of the study group are shown in The study conformed to the tenets of the declaration of Helsinki, and it was approved by the Office for Research Ethics Committees, Northern Ireland. All participants gave informed consent.rs12614 and rs641153 by PCR  followed by DNA sequencing using the forward primer. All samples showing the minor allele of rs12614 were confirmed using SNaPshot . Briefly, primer (5′-CAG GTG TGA CCA CCA CTC CAT GGT CTT TGG CC-3′) extension was performed on the PCR product using a fluorescent detection system. Data for the smoking status and CFH and HTRA1 haplotypes of these participants were available from our previous studies [DNA was extracted from peripheral blood leucocytes or frozen buffy coat samples. We performed SNP genotyping of  studies .PLINK v1.07 [PASW v18. The numbers of copies  of each CFH haplotype and CFB codon 32 variant carried by an individual were included as variables, omitting a reference haplotype from each gene. CFH haplotypes were tagged by rs6677604, rs3753396, rs419137, and rs2284664 , with haplotype 3 omitted as the reference. The heterozygous or homozygous state for the HTRA1 risk allele  was included, with homozygosity for the major allele  omitted as the reference. Current smoking and past smoking were included in the model as binary variables, with having never smoked omitted as the reference. Associations between disease status and allele frequency in the studies performed by ourselves and other groups were investigated using Pearson’s χ2 test in Epi-Info v6. To enable comparison of data sets from other groups, we used their published data, multiplying allele frequencies by the number of cases or controls to give the allele count if these were not published. For the meta-analysis, we compared the counts of 32W and 32Q alleles to the reference variant, 32R. Statistical significance was accepted at p<0.05 for all tests.Deviation from the Hardy–Weinberg equilibrium was investigated by use of the exact method of Wigginton et al.  as used NK v1.07 . Logistirs641153 and rs12614 were in Hardy–Weinberg equilibrium in cases and controls. The genotyping rate was 99.7%. CFB genotype data were available for 618 participants.Both −5), and the 32W variant was associated with an odds ratio of 0.64 (p=0.04).The 32Q and 32W variants of CFB were found to be protective against the disease when compared to the 32R variant . The 32QCFH haplotypes 1 and 2, which carry the Y402H risk allele [CFH haplotype 5, which carries the deletion of CFHR3 and CFHR1 [HTRA1 risk allele was associated with increased risk, particularly in homozygosity (odds ratio 25.04). Past and current smoking were associated with increased risk  compared to having never smoked.The 32Q and 32W variants of CFB were found to be protective against AMD when smoking status and other known genetic risk loci were integrated into a logistic regression . The grak allele , were asnd CFHR1 , was proThe 32Q variant of CFB was significantly associated with protection from AMD in all of the studies that also reported data for 32W . The 32Wrs12614 in CFB (32W) and decreased risk of neovascular AMD has not been reported previously. The study by Montes et al. [rs641153) was associated with a more extreme reduction in complement activation relative to 32R. We investigated the hypothesis that the genetic variant causing 32W was associated with protection from AMD, and found this to be the case when compared to 32R by Pearson’s χ2, by logistic regression with rs641153 and by logistic regression that included other known risk factors for AMD. The two other studies of neovascular AMD  showed This association has not been detected previously. This discord may reflect chance findings of either our study or the studies of others. However, combined analysis of all available data supports the conclusion that CFB 32W is associated with moderate protection against neovascular AMD, a finding that is in keeping with the study by Montes et al., which showed decreased complement activation relative to CFB 32R .Our study provides further evidence that an individual’s risk of AMD is affected by gene polymorphisms that affect the function and interaction of the alternative complement pathway proteins.


Several studies have reported the association between methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms and nonsyndromic cleft lip with or without palate (NSCL/P) in Asian populations. However, findings have been conflicting. In order to investigate the association, a meta-analysis was performed.We searched Pubmed, MedLine and EmBase database to selected eligible studies. The pooled odds ratios (ORs) with 95% confidence intervals (95%CIs) were calculated using fixed effects model or random effects model to assess the association between MTHFR polymorphisms and NSCL/P in both Asian children and mothers.Finally, nine case-control studies were included. Overall, the MTHFR C677T polymorphism and NSCL/P showed pooled ORs (95%CI) of 1.41(1.23–1.61) in Asian children, and 1.70(1.19–2.42) in Asian mothers. Subgroup analyses by geographical locations further identified the association in Eastern Asian children, Western/Central Asian children and mothers, but not in Eastern Asian mothers. However, no significant relationship between MTHFR A1298C polymorphism and NSCL/P was found in this meta-analysis.The MTHFR 677T allele was associated with an increased risk of NSCL/P in Asian populations. Nonsymdromic cleft lip with or without palate (NSCL/P) is one of the most common congenital malformations of the head and neck area in the world, occurring in approximately 1 in every 700 live births Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the metabolism of folate. This enzyme irreversibly catalyzes the reaction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which is the primary circulating form of folate The first report evaluating the role of MTHFR gene polymorphisms in the development of NSCL/P was conducted by Tolarova et al.(1998) Eligible literatures were screened from PubMed, MedLine and EmBase database . We used the following keywords and subject terms: “methylenetetrahydrofolate reductase” or “MTHFR” and “cleft lip” or “cleft palate”. Furthermore, we manually searched references in the eligible articles. There was no language limitation. And the search results were limited to humans.Inclusion criteria were showed as following: (1) evaluation the association between MTHFR C677T and/or A1298C polymorphisms and NSCL/P risk in Asian populations; (2) case-control studies; (3) the frequencies of all genotype distribution or other available data for estimating the OR (95% CI). If overlapping cases or controls were presented in multiple studies, the most recent publication or the largest study was included. The exclusion criteria were: (1) not using a case-control study design; (2) had no detailed data on genotype distribution; (3) not in Asian populations.The following information was carefully and independently collected from each eligible study by two authors: first author's name, year of publication, country, population of cases and controls, genotyping method, and numbers of each genotype. The difference was settled by reaching an agreement among all authors.2 values were used to quantify the percentage of the total variation among studies when heterogeneity was assessed. The I2 value ranged from 0 to 100%. 25%, 50%, 75% expressed low, moderate, high heterogeneity, respectively. When I2<50%, a fixed effects model was applied to estimate the pooled results. Otherwise, the random-effect model was used.To assess the study quality, Hardy–Weinberg equilibrium (HWE) in controls in each study was calculated by chi-squared test. P value<0.05 was considered a departure from HWE. The associations between MTHFR C677T and A1298C gene polymorphisms and NSCL/P risk were estimated by the odds ratios (ORs), together with the 95% confidence interval (95%CI). The significance of the pooled OR was determined by the Z test, with P<0.05 considered significant. Heterogeneity between studies was assessed by Q test. If P<0.05, the heterogeneity was considered statistically significant. The IPublication bias was investigated visually in a funnel plot of log (OR) against its standard error (SE). An asymmetric plot suggested possible publication bias. And the degree of asymmetry was assessed by Egger's test. Sensitivity analysis was performed by omitting each study in turn to assess the results stability.Software STATA version 11.0 was used for all analyses.A flow chart summarizing the process of study selection is shown in 2 = 42.3%), homozygote comparison (TT vs. CC: I2 = 28.3%) and recessive comparison (TT vs. CC/CT: I2 = 17.4%) in Asian children, and recessive comparison (TT vs. CC/CT: I2 = 49.8%) in Asian mothers. Overall, the variant T allele of MTHFR C677T increased risk of NSCL/P, when compared with the wild-type C allele. The OR (95%CI) was 1.41, heterozygote comparison (AC vs. AA: I2 = 76.0%) and dominant comparison (AC/CC vs. AA: I2 = 74.8%) in Asian children. However, no significant associations between MTHFR A1298C polymorphism and NSCL/P were found under any of genetic model, even in the subgroup analyses by geographical location, neither in Asian children nor in Asian mothers.For MTHFR A1298C polymorphism, the main results were shown in Elimination of each study made no qualitative difference on the pooled OR values, which indicated that the final results of the meta-analysis were stable. The publication bias of the included studies was assessed by the Funnel plot and Egger's test. The shapes of Funnel plot in each genotype comparison indicated no obvious asymmetry . And EggIn this meta-analysis, we investigated the association between MTHFR C677T and A1298C polymorphisms and NSCL/P, with a total of nine case-control studies included. The pooled results indicated that there was an obvious association between MTHFR C677T polymorphism and NSCL/P risk in Asian children under all models: allele contrast (T vs. C), homozygote (TT vs. CC), heterozygote (CT vs. CC) and dominant (TT/CT vs. CC) models, except for recessive (TT vs. CT+CC) model. Meanwhile, the variant T allele of Asian mothers was significantly associated with a 1.70-fold increased risk of having a NSCL/P offspring. Subgroup analyses by geographical location further identified this association in Eastern Asian children, Western/Central Asian children and mothers, but not in Eastern Asian mothers. Regarding MTHFR A1298C polymorphism, the pooled results revealed that A1298C was not related to children's or mothers' NSCL/P susceptibility under any of the genetic model, even in the subgroup analyses by geographical location. The above results suggested that MTHFR C677T polymorphism was a risk factor in the development of NSCL/P in Asian populations.As far as we know, there has been four published meta-analyses regarding MTHFR polymorphisms and NSCL/P risk. According to Verkleij-Hagoort et al.(2007) Some limitations of this meta-analysis should be acknowledged. Firstly, the sources of control among the studies were different from each other. Some studies were population-based studies, and others were hospital-based studies. Secondly, our results were based on unadjusted OR values that lack the original data from the eligible studies, which could lead to relatively weak power to estimate the real relationship. Thirdly, our analyses were based on single-factor estimates, which overlooked the interactions of gene-gene and gene-environment in the development of NSCL/P. Finally, the sample size was relatively small to investigate the association between MTHFR C677T and A1298C polymorphisms and NSCL/P risk.In conclusion, our meta-analysis suggested that MTHFR C677T polymorphism was related with an increased NSCL/P risk in Asian populations. In the future, large sample studies should be warranted to investigate the association between MTHFR C677T and A1298C polymorphisms and NSCL/P, and to examine the potential gene-gene and gene-environment interactions.Checklist S1(DOC)Click here for additional data file.


ATP-binding-cassette-member A3 (ABCA3) are related to severe chronic lung disease in neonates and children, but frequency of chronic lung disease due to ABCA3 mutations in the general population is unknown. We tested the hypothesis that individuals heterozygous for ABCA3 mutations have reduced lung function and increased risk of COPD in the general population.Mutations in ABCA3 gene. We genotyped the entire Copenhagen City Heart study  to assess the clinical importance of these mutations. To validate our findings we genotyped an additional 54,395 individuals from the Copenhagen General Population Study.We screened 760 individuals with extreme pulmonary phenotypes and identified three novel  and four previously described mutations  in the 1 % predicted compared with noncarriers (t-test: p = 0.008), and an increased odds ratio for COPD of 1.9 (95% CI: 1.1-3.1). In contrast, the A1086D mutation was associated with increased FEV1 % predicted (p = 0.03). None of the other ABCA3 mutations associated with lung function or COPD risk in the Copenhagen City Heart Study. In the larger Copenhagen General Population Study, and in the two studies combined, E292V heterozygotes did not have reduced lung function or increased risk of COPD (p = 0.11-0.98), while this was the case for the positive controls, surfactant protein-B 121ins2 heterozygotes and α1-antitrypsin ZZ homozygotes.In the Copenhagen City Heart Study individuals heterozygous for E292V had 5% reduced FEVOur results indicate that partially reduced ABCA3 activity due to E292V is not a major risk factor for reduced lung function and COPD in the general population. This is an important finding as 1.3% in the Danish population has partially reduced ABCA3 function due to E292V. Pulmonary surfactant is a thin lipid membrane that covers the alveoli and lowers surface tension, thereby preventing collapse of the alveoli at end-expiration. Pulmonary surfactant consists of phospholipids and proteins mainly synthesized by alveolar type II cells. The pulmonary surfactant is assembled and stored in the alveolar type II cell in lamellar bodies, which are subsequently released to the alveoli by exocytosis ,2. The lABCA3 gene have been related to deficiency of pulmonary surfactant leading to neonatal lung disease and chronic lung disease in children [ABCA3 mutations in the general population is unknown. At present more than 150 disease-associated mutations have been identified [ABCA3, E292V, is associated with partially impaired ABCA3 function [ABCA3 variants may also be at increased risk of emphysema/COPD. Given this, ABCA3 variants, and in particular E292V, could play an important role on development of common pulmonary disorders in the general population.Recessive mutations in the children ; howeverfunction  and withfunction . This mufunction  and may function ,9 and cofunction ,10. Becafunction , individABCA3 variants have reduced lung function and increased risk of COPD in the general population. To test this, we first screened 760 adults with extreme lung phenotypes in the Copenhagen City Heart Study to identify ABCA3 variants that could potentially be associated with lung disease in the general population. Following this we genotyped the entire Copenhagen City Heart Study  for variants identified by the initial screening to test whether ABCA3 heterozygotes have reduced lung function or increased risk of COPD. When a positive association was observed, we genotyped an additional population-based study, the Copenhagen General Population Study  in an attempt to reproduce the finding. As positive controls for reduced lung function due to genetic background, we used surfactant protein-B 121ins2 heterozygosity and α1-antitrypsin ZZ homozygosity [In this study we hypothesise that individuals heterozygous for zygosity .The Copenhagen City Heart Study is a prospective general population study of individuals selected based on the Central Population Register Code to reflect the adult Danish population aged 20-80+ years ,14. The The Copenhagen General Population Study is an ongoing population-based cohort study initiated in 2003 ,15. At t1 and FVC were determined with a dry wedge spirometer  in the Copenhagen City Heart Study, and with EsyOne Spirometer  in the Copenhagen General Population Study. Algorithms for calculation of FEV1 % predicted and FVC% predicted were made using multiple regressions with age and height as covariates on all individuals for men and women separately. COPD was defined as FEV1/FVC < 0.7 and FEV1 < 80% of predicted (GOLD stages 2–4) [ABCA3 associated with lung disease, we performed resequencing of the ABCA3 gene in an extreme risk population in the Copenhagen City Heart Study , lowest FEV1 % predicted among smokers (n = 122), and highest FEV1 % predicted in the population (n = 140) [1 % predicted and selected those 140 individuals who had the highest FEV1 % predicted. We next selected the 122 individuals with the lowest FEV1 % predicted among current smokers and the 118 individuals with the lowest FEV1 % predicted among exsmokers and nonsmokers to fill out the DNA plate in full.FEVges 2–4) ; if thisn = 140) . We ensuABCA3 and the intron-exon boundaries. Mutational screening analysis of the amplicons was performed by LightScanner, a high resolution DNA melting curve analysis technique for variant detection [Thirty PCR fragments were amplified, covering all 30 protein coding exons (exons 4–33) of etection . PCR fra1 % predicted, FVC % predicted, and FEV1/FVC were tested by Student’s t-test or ANCOVA. Odds ratios for spirometry defined COPD according to genotype were determined by a logistic regression model adjusted for age, sex, and packyears of tobacco smoked. As positive controls for reduced lung function and increased COPD risk due to genetic background, we used surfactant protein-B 121ins2 heterozygosity and α1-antitrypsin ZZ homozygosity [Statistical analyses were performed using STATA version 10.0. A two-sided P < 0.05 was considered significant. Main effects of genotype and statistical interaction between genotype and smoking in predicting FEVzygosity . We util1 % predicted did not differ in sex or age from those without an extreme lung phenotype.Clinical characteristics of the individuals with extreme lung phenotypes are listed in Additional file ABCA3 gene identified a total of 55 gene variations .We genotyped the entire Copenhagen City Heart Study for all seven 1 % predicted (t-test:p = 0.008), 3% reduced FVC %predicted (p = 0.04) and 0.02 reduced FEV1/FVC (p = 0.03), compared with wildtypes  or a 214 ml reduction of FEV1 in a 40 year old male (height 180 cm).Individuals heterozygous for E292V had 5% reduced FEV1 % predicted (p = 0.03) and FVC % predicted (p = 0.008) compared with wildtypes, whereas the FEV1/FVC ratio showed no statistical significant difference (p = 0.51). Because this result was based on a small number of heterozygous individuals (n = 4), it is likely that this could be a chance finding. Furthermore, due to the improved spirometry observed, this mutation is unlikely to cause lung disease. Thus we did not attempt to reproduce this association in the Copenhagen General Population Study.In contrast, A1086D heterozygotes had increased FEV1 % predicted, FVC % predicted or FEV1/FVC (p≥0.19). We sought to examine whether a combination of multiple variations in ABCA3 were associated with reduced lung function and COPD risk; however, only a single individual in the Copenhagen City Heart Study was heterozygous for more than one of the seven variants. This individual was compound heterozygous for P766S and S1262G and was not identified among the extreme phenotypes group. This person had FEV1/FVC of 0.71 and FEV1 % predicted of 83%. If correction for multiple comparisons was performed in Figure 1/FVC in ZZ homozygotes would be of statistical significance.Individuals heterozygous for H86Y, A320T, P766S, S1262G, or R1474W did not differ from wildtypes in FEVsurfactant protein-B 121ins2 heterozygotes in Figure ABCA3 mutations associated with asthma in the Copenhagen City Heart Study (data not shown).Individuals heterozygous for E292V had a multivariate adjusted odds ratio for COPD of 1.9 (95% CI 1.1-3.1) compared with wildtypes in the Copenhagen City Heart Study  for the E292V variant. The genotype distribution did not differ from that in the Copenhagen City Heart Study , whereas FEV1 % predicted was reduced in the positive controls, surfactant protein-B 121ins2 heterozygotes and α1-antitrypsin ZZ homozygotes. In accordance with the results on pulmonary function, the multivariate adjusted odds ratio for COPD was not increased in E292V heterozygotes compared with wildtypes (odds ratio 1.10 (0.83-1.46)), while surfactant protein-B 121ins2 heterozygotes and α1-antitrypsin ZZ homozygotes had increased odds ratios for COPD of 2.8 (1.0-8.1) and 6.6 (1.7-26) , whereas FEV1 % predicted was reduced in the positive controls, surfactant protein-B 121ins2 heterozygotes and α1-antitrysin ZZ homozygotes , 6% reduced FVC % predicted (p = 0.01), and 6% reduced FEV1/FVC (p = 0.00007) compared with wildtypes. Corresponding lung function reductions for α1-antitrypsin ZZ homozygotes were 24% (p = 0.0001), 8% (p = 0.10), and 19% (p = 0.0001). Among nonsmokers, lung function did not differ in surfactant protein-B 121ins2 heterozygotes and α1-antitrysin ZZ homozygotes versus wildtypes (p≥0.43).Finally, to maximize our statistical power, we combined the Copenhagen City Heart Study and Copenhagen General Population Study. In this analysis, we found that FEVs Figure . We alsosurfactant protein-B 121ins2 heterozygotes were 2.1 (1.7-3.8), 1.4 (0.3-5.9), and 2.4 (1.2-4.8), and for α1-antitrypsin ZZ homozygotes 6.0 (2.2-16), 4.8 (0.3-40), and 11 (3.1-42). These results were in accordance with the results observed for lung function for these gene variants. We had 80% power to exclude odds ratios for COPD for E292V heterozygotes of 1.3, for surfactant protein-B 121ins2 heterozygotes of 2.1, and for α1-antitrypsin ZZ homozygotes of 4.0.Individuals heterozygous for E292V had multivariate adjusted odds ratios for COPD of 1.2 (0.96-1.57) among all subjects, of 1.1 (0.6-2.1) among nonsmokers, and of 1.2 (0.95-1.63) among smokers, respectively, compared with wildtypes Figure . CorrespABCA3 variants have reduced lung function and increased risk of COPD in the general population, we screened 760 individuals with extreme lung phenotypes for genetic variations in the ABCA3 gene and identified three novel  and four previously described variations . We next genotyped the entire Copenhagen City Heart Study  to assess the clinical importance of these seven mutations. We found that E292V heterozygotes had reduced lung function and increased risk of COPD, whereas the novel A1086 mutation was associated with increased lung function. To validate the findings for E292V we genotyped an additional 54,395 individuals from the Copenhagen General Population Study. In this larger cohort, and in the two study cohorts combined, we found that E292V heterozygotes did not have reduced lung function or increased risk of COPD. Supporting this overall negative result, the estimates for the positive controls, surfactant protein-B 121ins2 heterozygotes and α1-antitrypsin ZZ homozygotes, were relatively stable throughout the studies and there was no evidence of selection bias against E292V in any of the two cohorts.To test whether individuals heterozygous for surfactant protein-C[ABCA3[surfactant protein-B could be risk factors of additionally impaired surfactant function in E292V heterozygotes; although the latter risk factors are probably less prevalent in the general population as compared with tobacco smoking. Because participants in the Copenhagen City Heart Study smoked more tobacco than participants from the Copenhagen General Population Study we speculate that the difference in smoking habits between the two cohorts could partly contribute to the different results observed for E292V in the two cohorts. When statistical power, however, was maximised using the Copenhagen General Population Study or the two studies combined, E292V heterozygotes did not differ from wildtypes in lung function or COPD risk. This is an important finding as 1.3% in the Danish general population has partially reduced ABCA3 function due to E292V, and since this variant has been linked previously with severe chronic lung disease in heterozygous and compound heterozygous E292V carriers [The phenotype in E292V carriers was previously reported to range from minimal changes on lung biopsy and no symptoms to severe fatal lung disease ,9. It isprotein-C,25, ABCAn-C[ABCA3, and surcarriers -10.1 % predicted and FVC % predicted. However, the small number of A1086D heterozygotes (n = 4) makes this finding highly insecure. In addition, if correction for multiple comparisons was performed none of the results for A1086D and E292V in the Copenhagen City Heart Study would be of statistical significance. The second novel mutation, H86Y, is situated in the first extracellular loop of ABCA3. Another mutation in this loop, L101P, has been shown to affect ABCA3 protein folding leading to retention of ABCA3 in the endoplasmatic reticulum and subsequent ER stress and apoptosis [Besides E292V, we identified six other variants of potential relevance to ABCA3 function. However, none of these mutations were associated with lung function or risk of COPD, except for the novel A1086D mutation. This variant associated with increases in FEVpoptosis ,28, howepoptosis . Howeversurfactant protein-B 121ins2 heterozygotes had reduced FEV1 % predicted and increased risk of COPD. This observation is novel and extends our previous finding of reduced lung function and elevated COPD risk among 121ins2 heterozygous smokers [surfactant protein-B 121ins2 heterozygotes in this study was still driven by an effect among smokers, while nonsmokers were unaffected  and the Copenhagen General Population Study (CGPS).Click here for fileFigure S2.Click here for fileFigure S3.Click here for file


Association between parity and colorectal cancer (CRC) risk has been investigated by several epidemiological studies but results are controversial, yet a comprehensive and quantitative assessment of this association has not been reported so far.Relevant published studies of parity and CRC were identified using MEDLINE, EMBASE and Web of Science databases through end of April 2013. Two authors independently assessed eligibility and extracted data. Eleven prospective studies reported relative risk (RR) estimates and 95% confidence intervals (CIs) of CRC risk associated with parity. We pooled the RR from individual studies using fixed- or random-effects models and carried out heterogeneity and publication bias analyses.Q = 9.04, P = 0.443, I2 = 0.5%). Likewise, no significant association was yielded for the highest vs. lowest parity number , with moderate heterogeneity . Dose-response analysis still indicated no effect of parity on CRC risk and the summary RR of per one livebirth was 0.99 (95% CI: 0.96–1.02), with moderate of heterogeneity . Similar results were observed among all the subgroup analyses. No evidence of publication bias and significant heterogeneity between subgroups were detected by meta-regression analyses.The summary RR for the ever parity vs. nulliparous was 0.95 (95% CI: 0.88–1.02), with no heterogeneity (Results of this dose-response meta-analysis of prospective studies found that there was little evidence of an association between parity and CRC risk. Colorectal cancer (CRC) is the second most commonly diagnosed cancer and third leading cause of cancer death worldwide in females, with over 570,100 new cases and 288,100 cancer deaths in 2008, which constituted a significant proportion of the global burden of cancer morbidity and mortality Reproductive factors, such as pregnancy, age at menarche, and age at menopause, have been used as surrogate markers for lifetime exposure to endogenous estrogens We performed a comprehensively literature search to April 2013 using MEDLINE, EMBASE, and Web of Science databases for epidemiological studies evaluating the association between parity  and the risk of CRC. The search was limited to studies of humans using the following search key words and medical subject heading terms: (parity OR pregnancy OR livebirth OR reproductive OR reproduction OR reproductive factors) AND  AND (cancer OR neoplasm OR carcinoma OR tumor). We also reviewed the references of all included studies for additional publications. This systematic review was planned, conducted, and reported in adherence to standards of quality for reporting meta-analyses Published studies were included if they 1) used a prospective study design; 2) evaluated the association between parity and CRC risk; 3) presented relative risk (RR) or hazard ratio (HR) estimates with 95% confidence intervals (CI), standard errors (SE) or data necessary to calculate these. When multiple publications from the same study were available, we used the publication with the largest number of cases and most applicable information. The detailed steps of our literature search are shown in For each eligible study, two investigators (H-BG and Q-JW) independently performed the eligibility evaluation, data abstraction, and quality assessment. The disagreements were discussed and resolved by consensus. Data abstracted from each study were: author list, year of publication, study region, study sample size (number of cases and cohort size), range of follow-up of studies, exposure and outcome assessment including parity and the number of parity categories, study-specific adjusted estimates with their 95% CIs for the ever parous versus nulliparous, highest versus lowest number (including nulliparous) of parity, and factors matched by in the design or adjusted for in data analysis. If multiple estimates of the association were available, we abstracted the estimate that adjusted for the most covariates. If no adjusted estimates were presented, we included the crude estimate. If no estimate was presented in a given study, we calculated it and its 95% CI according to the raw data presented in the article.To assess study quality, a 9-star system on the basis of the Newcastle-Ottawa Scale The study-specific adjusted RRs were used as the measure of association across studies. Because the absolute risk of CRC is low, we assumed that estimates of risk, rate or hazard ratios from prospective studies were all valid estimates of the RR and we therefore report all results as the RR for simplicity. For one study that did not use the category with the lowest number of parity as the reference, we used the effective count method proposed by Hamling et al For the dose-response analysis, we used the method proposed by Greenland et al Q statistic, where P<0.1 was indicative of statistically significant heterogeneity, and the I2 statistic. The summary estimate based on the fixed-effects model We evaluated heterogeneity of RRs across studies by using the Cochrane P-value less than 0.05 for Egger’s or Begg’s tests was considered representative of significant statistical publication bias. Statistical analyses were performed with Stata . P-values were two sided with a significance level of 0.05.Publication bias was evaluated via Egger’s linear regression Study-specific quality scores are summarized in Q = 9.04, P = 0.443, I2 = 0.5%) (P for bias = 0.739) or with Begg’s test (P for bias = 0.929) and no asymmetry was observed in the funnel plots when inspected visually (data not shown).Eight cohort  = 0.5%) . There wQ = 17.48, P = 0.094, I2 = 37.1%) (P for bias = 0.734) or with Begg’s test (P for bias = 0.891) and no asymmetry was seen in the funnel plots when inspected visually (data not shown).Ten cohort = 37.1%) . There wQ = 13.54, P = 0.196, I2 = 26.1%) after omission of the study by Martínez et al Q = 14.69, P = 0.100, I2 = 38.7%) after omission of the study by Broeders et al Q = 10.16, P = 0.254, I2 = 21.3%).In a sensitivity analysis, we sequentially removed one study at a time and re-analyzed the data. The 11 study-specific RRs of the number of parity ranged from a low of 1.00  (P = 0.656), Begg’s test (P = 0.458) and visual inspect of the funnel plot (data not shown). Additionally, we found no evidence that the number of parity was associated with colon cancer, rectal cancer, and the subsite of colon cancer (Six cohort = 57.6%) . Publican cancer .Q = 9.13, P = 0.166, I2 = 34.3%) when Tsilidis et al Q = 8.37, P = 0.137, I2 = 40.3%) when Broeders et al Q = 10.20, P = 0.177, I2 = 31.4%), similar to the original analysis including all studies. Similarly, we also explored the effect on the results of excluding three studies which referred to one livebirth as the lowest category of parity number and the summary RR was 0.98 .In a sensitivity analysis excluding one study at a time, the summary RR for CRC ranged from 0.98 Click here for additional data file.


Furthermore, we performed a meta-analysis for effects of rs9514089 on susceptibility for cholelithiasis in three independent cohorts .Here we assessed the effects of rs9514089 with gallstone risk, serum lipid parameters and BMI in the Sorbs and in the meta-analysis of all three cohorts (p > 0.05). There was an effect trend in the subgroup of lean subjects but based on different effect directions in the three cohorts there was no significant association in the meta-analysis.There was no significant association of SLC10A2 in human gallstone development and lipid metabolism.We were not able to replicate the effect of rs9514089 on gallstone risk in the Sorbs. Further analyses in larger cohorts are required to finally assess the role of genetic variants in  SLC10A2  as a novel susceptibility gene for cholelithiasis in humans [SLC10A2 encodes the cholangiocyte bile salt transporter protein whose expression is reduced by a lithogenic diet in mice [The pathogenesis of gallstone disease is complex and a variety of environmental predisposing factors such as obesity and rapid weight loss, nutrition, certain medications and number of pregnancies have been identified -6. Howevn humans . SLC10A2 in mice  and medi in mice ,22. ASBT in mice  and to p in mice . It has  in mice ,26. The  in mice ,26.rs9514089 mapping within the SLC10A2 locus is a genetic determinant of gallstone disease, expressing gender and weight specificity with higher risk observed in men and in normal-weight subjects [rs9514089 on gallstone risk and related phenotypes of the metabolic syndrome in the self-contained population of Sorbs and performed a meta-analysis for effects of rs9514089 on susceptibility for cholelithiasis in the three independent cohorts .The recent study by Renner et al. suggested that a single nucleotide polymorphism (SNP) subjects . Since tAll subjects are part of a sample from an isolated population in Germany, the Sorbs ,28.®Insulin assay . Serum glucose was measured by the hexokinase method . Cases were defined as subjects with gallstones in the ultrasonic testing or those who underwent cholecystectomy due to symptomatic gallstones. Subjects with ultrasonic exclusion of gallstones were included as controls in this study. There were no other specific inclusion and exclusion criteria. A summary sample description is presented in Table At present, about 1000 Sorbs are enrolled in the study. Sampling comprised unrelated subjects as well as families. Extensive phenotyping included standardised questionnaires for past medical history and family history, collection of anthropometric data ) and a 75g-Glucose-tolerance-test (OGTT). Insulin was measured with the AutoDELFIAThe study was approved by the ethics committee of the University of Leipzig and all subjects provided written informed consent before taking part in the study.rs9514089 was performed using the TaqMan allelic discrimination assay (Assays-on-Demand (TM), SNP Genotyping Products; Applied Biosystems, Inc.) on an ABI PRISM 7500 sequence detector (Applied Biosystems Inc.) according to the manufacturer's protocol. The genotype distribution was consistent with Hardy-Weinberg equilibrium . Genotyping success rate was >99%, and duplicate genotyping concordance was 100%.Genotyping of Standard descriptive and comparative statistics (ANOVA) were used to characterize and compare clinical parameters in cases and controls Genetic associations were assessed by linear or logistic regression using an additive model of inheritance unless stated otherwise, and adjusted for age, gender and BMI. All effect directions were standardized to the minor allele.To obtain the combined effect of the three cohorts we performed a meta-analysis using the metan command in STATA based on the estimated effect sizes of each study and their 95% confidence intervals. The meta-analysis was performed in a fixed effect model by using the Mantel-Haenszel method.All statistical analyses were performed using the SPSS 15.0.1 software package  and STATA version 9.0, .2 variant rs9514089 tended to be associated with gallstones , whereas there was no effect in the group with BMI > 26 kg/m2 (p = 0.52)  = 38, N(GA carriers) = 124, N(AA carriers) = 131), but would not sustain correction for multiple testing.In our study, which includes 826 controls and 183 patients with gallstones, rs9514089 did not show any significant effect on gallstone prevalence, neither in the additive nor in the recessive or dominant mode of inheritance . In the subgroup of subjects with BMI ≤ 26 kg/mrs9514089 with serum parameters of lipid metabolism  in the subgroup of Sorbs without lipid-lowering medication , the SNP tended to be associated with birth weight (p = 0.03). Gallstone carriers were characterized by increased age, BMI, fat mass, waist- and hip circumference  . Also the stratified analyses and assessment of different genetic models did not reveal any consistent significant effect on gallstone risk  and assuming a recessive mode of inheritance. Although there was no significant difference in minor allele frequencies between the three cohorts the risk allele was the G allele in the Stuttgart and Aachen cohorts but the A allele in the Sorbs. This effect is most likely driven by the small samples sizes in each genotype group in the cohorts. Furthermore, gene-environmental interactions based on factors such as eating behavior and nutrition composition as well as gene-gene interactions should be taken into account. In our study the meta-analysis of all three cohorts did not indicate any significant effect of the rs9514089 genotype on gallstone risk. The Sorbs and the Aachen cohort showed a trend of association in the subgroup of non-obese individuals indicating a possible relationship between the SNP effect and obesity as suggested by Renner et al. [Recently, n humans . The datr et al. . This wor et al. ,26. AlsoSLC10A2 genotype and cholelithiasis was observed in the non-obese subgroup, even though the SNP effect could only be detected in women. This may be due to skewed gender distribution in favor of women and thereby greater statistical power for the female subset. The differences between the populations do not necessarily represent population specific effects of the gene variant. Consistent with the effect in the entire cohort the effect direction also in this subgroup was opposite in the Sorbs compared with the Aachen cohort. This could be attributed to population specific environmental factors or a different phenotype distribution in each population or it could be simply due to the lack of power based on the small samples sizes of the cohorts.In the Sorbs a nominal association of the rs9514089 on serum total cholesterol [Furthermore, our meta-analysis did not confirm the reported effects of lesterol . HoweverSLC10A2 in human gallstone development and lipid metabolismThe effect of rs9514089 genotype on gallstone risk was not replicated in the Sorbs. Further analyses in larger cohorts are required to finally assess the role of genetic variants in The authors declare that they have no competing interests.AT performed the phenotyping in the Sorbs, carried out the statistical analysis and drafted the manuscript. OR, SH, FL and EFS performed the genotyping and statistical analyses in the cohorts from Aachen and Stuttgart. PK and JH carried out the molecular genetic studies in the Sorbs. FL and HW participated in the design of the study. AT, PK and MS conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/12/149/prepub


To accomplish a more precise estimation of the relationship, a meta-analysis was performed.Caspases are important regulators and executioners in apoptosis pathway and have been defined as either tumor suppressors or oncogenes. Polymorphisms in promoter and exon of caspase 9 polymorphisms and cancer risk from nine studies with 5,528 subjects for rs4645978, six studies with 2,403 subjects for rs105276 and two studies for rs4645981. Overall meta-analysis indicated that no evidence of an association between rs4645978 and cancers was found. Through the stratified analysis, statistically significant reduced cancer risks were observed among Caucasians  and prostate cancer. As for rs105276, Ex5+32G>A polymorphism was found with protective effect in overall meta-analysis  and Asians group. While for rs4645981, a statistically significant increase in risk of lung cancer was shown in Asians .We assessed published studies of the association between caspase 9 rs4645978 most likely contributes to decreased susceptibility to cancer in Caucasians and prostate cancer. The A allele of rs105276 might be a protective factor for cancer, especially for Asians. However, it seems that rs4645981 confers increased susceptibility to lung cancer in Asians.Our meta-analysis suggests that the  Evasion of apoptosis is considered to be one of the hallmarks of various human cancers c from mitochondria in response to a variety of cellular stress. Released cytochrome c interacts with apoptotic protease activating factor 1 (Apaf-1), procaspase-9 and deoxyadenosine triphosphate (dATP) to form a multiprotein complex called apoptosome. Once bound to the apoptosome, CASP9 is active, which subsequently triggers a cascade of effector CASPs CASP9, a member of the intrinsic pathway, plays a central role in the mitochondrial apoptotic pathway. The intrinsic or mitochondrial pathway is initiated by the release of cytochrome CASP9 gene have been reported in public databases (http://www.ncbi.nlm.nih.gov/SNP). It is identified some of these SNPs in the promoter or exon sequence of CASP9 as potential biomarkers , which can modulate susceptibility to cancer, such as those that occur in the lung CASP9 SNPs with cancer risks.Recently, several candidate single nucleotide polymorphisms (SNPs) in We carried out a search in Medline, Embase, Chinese National Knowledge Infrastructure (CNKI) and Wangfang databases, covering all papers published between 1991 and 2012, with a combination of the following keywords: “caspase 9/caspase-9/CASP9”, “polymorphism/single nucleotide polymorphisms/SNPs/gene susceptibility/genetic variation” and “cancer/tumor/carcinoma/neoplasia/neoplasm” (last search was updated on 10 Mar 2012). We checked potentially relevant publications by examining their titles and abstracts and all studies matching the eligible criteria were retrieved. Besides the database search, the bibliographies of the selected papers and reviews were also examined by hand.CASP9 polymorphisms and cancer risks, (b) use a case-control design, (c) sufficient published data for estimating an odds ratio (OR) with 95% confidence interval (CI). Accordingly, the following exclusion criteria were also used: (a) reviews and repeated literatures, (b) not offering the source of cases and controls and other essential information, (c) not designed as case/control or cohort studies.Studies included in the current meta-analysis had to meet all the following criteria: (a) evaluation of the CASP9 genotypes, respectively. Different ethnicity descents were categorised as Caucasian, Asian and Mix .Data were independently abstracted by two investigators (Xu and Li) using a standard protocol and data-collection form according to the criteria listed above. Differences among evaluators were resolved through discussion and rereading with the third investigator (Wu). The following information was extracted from each included study using a standardized data collection protocol : the surCASP9 polymorphism and cancer. The significance of the pooled OR was determined by the Z-test, and P<0.05 was considered as statistically significant. For CASP9 rs4645978, the meta-analysis examined the association between G allele and cancer risk compared with that for A allele (G vs A); homozygote GG was contrasted with AA and recessive (GG vs AA+AG) and dominant (AA+GA vs GG) models for allele G were also used, so did rs10525576 and rs4645981. Subgroup analyses were stratified by the study characteristics of racial descent and tumor site, respectively.OR corresponding to 95% CI was used to assess the strength of association between Q-test P-value>0.10 for the Q-test shows a lack of heterogeneity among the studies, then the pooled OR estimate of each study was calculated by the fixed-effects model (the Mantel-Haenszel method) P-value<0.05 was considered significant. Publication bias was assessed by visual inspection of funnel plots in which the standard error of log (OR) of each study was plotted against its log (OR). Funnel plot asymmetry was assessed by the method of Egger's linear regression test. The significance of the intercept was determined by the t-test  P-values.Heterogeneity analysis was checked by the chi-square-based CASP9 polymorphisms and cancer susceptibility were identified. During the extraction of data, 13 articles were excluded, because they did not provide allele frequencies needed for OR calculation, the contents were lack of control, their contents mainly associated with cancer therapy, or they studied fewer CASP9 SPNs , leaving 15 eligible articles CASP9 polymorphism, that is rs4645978 and rs4645981. Therefore, each group in both studies was considered separately for pooling analysis. The characteristics of selected studies are summarized in CASP9 rs4645978, six studies CASP9 rs4645978, there were six subjects of Caucasians Twenty-eight studies probing the relationship between Frequencies of alleles were calculated for controls from the corresponding genotype distribution. The variant alleles had different representations among controls of Asian descent  and Caucasian descent .CASP9 rs4645978, five kinds of genetic models did not produce significant association among 9 studies with relatively large heterogeneity (Pheterogeneity = 0.002–0.096). Through stratified analyses, the heterogeneity of the subgroup significantly reduced. In the stratified analysis by racial descent, statistically significantly decreased cancer risks were found among Caucasian for AG vs AA . No significant risks were found among Asians. Carriage of the AG of rs4645978 was found to be associated with protection from prostate cancer . No significant associations were observed in lung cancer and other subgroups.For CASP9 rs105276 polymorphism and the reduced risks of cancers . In the subgroup analysis by ethnicity, statistically significantly decreased cancer risks were found among Asians for allele contrast  and the recessive model .The overall OR with its 95% CI showed statistically association between the vs C: OR = 1.23, 95% CI = 1.07–1.42, Pheterogeneity = 0.399; the dominant model: OR = 1.22, 95% CI = 1.04–1.43, Pheterogeneity = 0.660). All data are shown in As far as rs4645981, T allele had an effect of increasing the risk of lung cancer in Asians  and −712C>T (rs4645981), were observed to alter the transcriptional activity of promoter. An analysis of potential transcription factor-binding sites has shown that the G-C haplotype of −1263A>G and −712C>T polymorphisms have significantly higher transcriptional activity than the A-T haplotype CASP9 in G or C carriers offer protection against the development of some cancers. Our study found a significantly protective effect of CASP9 G variant in rs4645978 in the subgroups of Caucasians and prostate cancers. Besides, the C allele in rs4645981 also might be a protective factor for lung cancer in Asians.In current study, we first summarized the data about the association of intrinsic initiator CASP9  polymorphism encodes for a glutamine to arginine amino acid change at codon 221 of the protein, located at the border of the helix region of CASP9CASP9 Ex5+32G>A increases apoptosome activity which provides a protection from various disorders as well as cancer. Due to limited statistical power because of relative small sample size, further evaluation was warranted to confirm these results.CASP9 rs4645978 A allele and rs1052576 G allele among controls of Asians and Caucasian appeared. In the subgroup analysis by ethnicity, we found the significant association only in one ethnic but not in the other, suggesting genetic diversity between different ethnicities.One important property of the gene polymorphisms is that their incidence can vary substantially among different racial populations. In this meta-analysis, significant differences in the prevalence of the In interpreting the results, some cautions should be applied. First, relatively large heterogeneity existed in rs4645978 meta-analysis. Through stratified analyses by tumor site and racial descent respectively, heterogeneity reduced significantly. Therefore, we presumed that the relatively large heterogeneity mainly results from differences of ethnicity and tumor types. Simultaneously, the heterogeneity might also be caused by the differences in the selection of controls, age distribution and lifestyle factors. Moreover, lack of the original data of the reviewed studies limited our further evaluation of potential interactions because the interactions between gene-to-gene, gene-to-environment, and even different polymorphic loci of the same gene may modulate cancer risk. Finally, a relatively limited number of studies and samples was analyzed for our assessment. This was in part because the selected original studies were performed with rather limited sample sets. Also, because the reports included in our meta-analysis were limited to those published in either English or Chinese, it is possible that some relevant published and unpublished studies, likely to have null results, were not included, possibly biasing the result, although the result for publication bias was not statistically significant.caspase 9 rs4645978 most likely contributes to decreased susceptibility to cancer in Caucasians and prostate cancer. For rs105276, the A allele might be a protective factor for cancer, especially for Asians. However, it seems that rs4645981 confers increased susceptibility to lung cancer in Asians.In summary, this meta-analysis supports that the Figure S1Funnel plot of publication bias in rs4645978 studies. Log OR is plotted versus standard error for each of studies in this meta-analysis. Each point represents a separate study for the indicated association in all comparing models.(DOC)Click here for additional data file.Figure S2Funnel plot of publication bias in rs105276 studies. Each point represents a separate study for the indicated association.(DOC)Click here for additional data file.Table S1ORs (95% CI) of sensitivity analysis for rs4645978 and rs105276.(DOC)Click here for additional data file.File S1MOOSE Checklist and Flowchart for the meta-analysis.(DOC)Click here for additional data file.


The findings suggested an association between hsa-mir-499 rs3746444 polymorphism and increased risk to breast cancer.MicroRNAs (miRNAs) are gene regulators involved in numerous diseases including cancer, heart disease, neurological disorders, vascular abnormalities and autoimmune conditions. Although hsa-mir-499 rs3746444 polymorphism was shown to contribute to the susceptibility of multiple genes to cancer, the data have yielded conflicting results. Therefore, this meta-analysis was performed to provide a comprehensive assessment of potential association between hsa-mir-499 rs3746444 polymorphism and cancer risk. In this meta-analysis, a total of 9 articles regarding 10 eligible case-control studies in English (including 6134 cases and 7141 controls) were analyzed. No significant association between hsa-mir-499 rs3746444 polymorphism and overall cancer risk was demonstrated. However, an increased risk was observed in the subgroup of breast cancer patients (G allele  MicroRNAs (miRNAs) are short (around 22-nt) non-coding single-stranded RNA molecules involved in both physiological and pathological processes, exerting their regulatory effects by suppressing translation or by inducing the cleavage of target RNA transcriptset al.Single nucleotide polymorphisms (SNPs), the most common type of sequence variations in the human genome, contribute to human phenotypic differencesWe conducted a computerized literature search of PubMed, EmBase databases and Chinese National Knowledge Infrastructure (CNKI) databases . The following search terms were used in isolation and combination with one another: “mir-499 or rs3746444” and “polymorphism or variant or mutation” and “cancer or carcinoma”. The search was limited to papers published in the English language. The related reference articles were reviewed to identify other potentially eligible publications. All studies matching the eligible criteria were included in our meta-analysis. Studies included in the meta-analysis met the following criteria: 1) articles on hsa-mir-499 rs3746444 polymorphism and cancer risk; 2) studies using a case-control design; 3) studies containing sufficient published data for the estimation of odds ratios (ORs) with their 95% confidence interval (CI).P value for Hardy-Weinberg equilibrium (HWE), respectively.Two investigators extracted necessary data from all eligible publications independently according to the inclusion criteria listed above. From each of the included articles the following information was abstracted: the name of first author, year of publication, country origin, ethnicity , cancer type, source of controls , genotyping methods, total number of cases and controls, the number of cases and controls with hsa-mir-499 rs3746444 polymorphism genotypes, G allele frequency in controls and Z test . The pooled ORs were calculated and used for comparisons between two homozygotes (GG vs AA), two heterozygotes (AG vs. AA), dominant models (GG+AG vs AA), recessive models (GG vs AG+AA) and the allele contrast models (G vs A), respectively. Subgroup analyses were also performed by cancer types , ethnicity and source of controls. Inter-study heterogeneity was estimated using a chi-square-based Q-test. The pooled ORs were analyzed using a random effects model (the DerSimonian and Laird method)P < 0.05) provided by the Q-test, and using a fixed-effects model (the Mantel-Haenszel method)P > 0.05) was found. The I2 (I2 = 100% × (Q-df)/Q) statistic was then used to quantitatively estimate heterogeneity, where I2 < 25%, 25-75% and >75% represent low, moderate and high inconsistency, respectivelyP < 0.05 was considered a significant publication bias)We first assessed HWE for the controls in each study. The strength of the association between polymorphism and cancer risk was assessed by ORs with 95% CIs. The statistical significance of the summary OR was determined by et al.Through an extensive search, 9 articles regarding 10 case-control studies in English (including 6134 cases and 7141 controls) met the inclusion criteria. In the study by Catucci P = 0.01) higher than that in Asian populations  , significantly . Similarly, insignificant effects were also found under the dominant and recessive model. vs A allele).As shown in vs A, OR = 1.10, 95% CI = 1.00-1.20; Pheterogeneity = 0.114; I2 = 53.9%) rather than in other cancer types , not in the hospital-based studies . In the subgroup analyses of cancer types, ethnicity and source of controls, heterogeneity was not observed any longer in breast cancer  and other cancer , in Caucasian populations , and in population-based  and hospital-based studies .There was significant heterogeneity in three genetic models  for the G vs A allele contrast model.We used Begg's funnel plot and Egger's test to assess the publication bias. The graphical funnel plots for the comparison of the G allele and the A allele appeared to be symmetrical  and cancer in population-based studies . Further larger, preferably prospective studies are required to evaluate the role of hsa-mir-499 rs3746444 polymorphism in cancer risk.In conclusion, this meta-analysis provided evidence that hsa-mir-499 rs3746444 polymorphism is not associated with cancer risk. However, stratified analysis indicated a significantly increased risk of breast cancer (G


MDM4 3′-untranslated region creates a miR-191 target site and results in decreased MDM4 expression. Therefore, we investigated the association between this polymorphism and esophageal squamous cell carcinoma (ESCC) risk as well as its biological function in vivo. Genotypes were determined in two independent case-control sets consisted of 1128 ESCC cases and 1150 controls from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. The impact of the polymorphism on MDM4 expression was examined with esophagus tissues. Our results demonstrated that the MDM4 rs4245739 AC and CC genotypes were significantly associated with decreased ESCC risk compared with the AA genotype in both case-control sets . Stratified analyses revealed that a multiplicative interaction between rs4245739 and smoking or drinking was evident . After detecting In vivo MDM4 mRNA expression, we found that the rs4245739 AC and CC genotype carriers had significantly decreased MDM4 expression in normal esophagus tissues compared with AA genotype carriers, indicating a consistent genotype-phenotype correlation. Our results elucidate that the MDM4 rs4245739 polymorphism contributes to susceptibility of ESCC and support the hypothesis that genetic variants, interrupting miRNA-mediated gene regulation, may modify cancer risk.As an oncoprotein, MDM4 plays a key part in P53 tumor suppressor pathway through negatively regulating P53 function. It has been reported that an rs4245739 A>C polymorphism locating in the  Esophageal squamous cell carcinoma (ESCC) is one of the most common and fatal cancers in the word, showing a relatively high morbidity in Asian, especially in China MDM4 is a bona fide oncogene in vivo cooperating with P53 As a tumor suppressor, P53 plays a pivotal role in maintaining genomic stability and controlling cell growth as well as apoptosis MDM4 that creates a putative target site for miR-191 MDM4 mRNA and protein levels among MDM4 rs4245739 A allele carriers in ovarian cancer and retinoblastoma P = 0.02] increased risk of recurrence and 5.5-fold  increased risk of tumor-related death compared with cases with AC or CC genotype MDM4 rs4245739 SNP may be also involved in ESCC development through impacting miR-191-mediated differential regulation of MDM4 expression. To test this hypothesis, we conducted a large case–control study of ESCC from two different regions of China. To validate the biological function of this SNP in vivo, we examined the association between its genotypes and MDM4 mRNA expression levels in normal and cancerous esophagus tissues.In a previous study, Wynendaele et al showed that an SNP (rs4245739 A>C) in the 3′-untranslated region (3′-UTR) of There were two case-control sets in the current study. (a) Jinan case-control set: 540 patients with ESCC from Shandong Cancer Hospital, Shandong Academy of Medical Sciences  and sex- and age-matched (±5 years) 550 controls. Patients were recruited between June 2009 and April 2012 at Shandong Cancer Hospital. Control subjects were randomly selected from a pool of 4500 individuals from a community cancer-screening program for early detection of cancer conducted in Jinan city during the same time period as the patients were collected. (b) Huaian case-control set: 588 ESCC patients from Huaian No. 2 Hospital  and sex- and age-matched 600 controls. Patients were consecutively recruited between January 2009 and February 2012 at Huaian No. 2 Hospital. Controls were cancer-free individuals selected from a community cancer-screening program  for early detection of cancer conducted in Huaian city during the same time period as the patients were collected. The diagnosis of all patients was histologically confirmed. Individuals who smoked one cigarette per day for over 1 year were considered as smokers. Subjects were considered as alcohol drinkers, if they drank at least once per week. Twenty-nine esophagus normal tissues adjacent to the tumors and twenty nine paired ESCC tissues were obtained from surgically removed specimens of patients in Huaian No. 2 Hospital. The normal tissues sampled at least 2 cm away from the margin of the tumor. Part of the two case-control sets and the tissue samples has been reported previously MDM4 rs4245739 A>C genotypes were determined using PCR-based restriction fragment length polymorphism (RFLP). During genotyping, the primers used for amplifying DNA segments with the SNP site (the mismatch base is underlined) were 5′-AAGACTAAAGAAGGCTGGGG-3′ and CC-3′5′-TTCAAATAATGTGGTAAGTGA. The PCR was performed with a 25 µL 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 rTaq DNA polymerase (TaKaRa), 1×reaction buffer, and 1.5 mmol/L MgCl2. The PCR profile consisted of an initial melting step of 2 minutes at 95°C, followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 58°C, 30 seconds at 72°C, and a final elongation step of 10 minutes at 72°C. Restriction enzyme MspI (New England Biolabs) was utilized to distinguish the rs4245739 A>C genotypes. A 15% random sample was reciprocally tested by different person, and the reproducibility was 99.9%. In addition, a 5% random sample was also examined by Sanger sequencing, and the reproducibility was 100%.MDM4 mRNA levels in normal and cancerous esophagus tissues as described previously MDM4 and GAPDH as an internal reference gene was carried out using the ABI 7500 real-time PCR system in triplicates. The primers used for MDM4 were 5′-CTACCGAGTGTCTGTCTAAG-3′ and 5′-TCCTGGGTGTTTGTATTT-3′; and for GAPDH were 5′-AACAGCGACACCCATCCTC-3′ and 5′-CATACCAGGAAATGAGCTTGACAA-3′.SYBR-Green real-time quantity PCR method was used to examine 2 test was used to examine the differences in demographic variables and genotype distributions of MDM4 rs4245739 polymorphism between ESCC patients and controls. Associations between MDM4 rs4245739 genotypes and risk of the development of ESCC were estimated by OR and their 95% CIs computed using unconditional logistic regression model. All ORs were adjusted for age and sex, where it was appropriate. We tested the null hypotheses of multiplicative gene-covariate interaction and evaluated departures from multiplicative interaction models by including main effect variables and their product terms in the logistic regression model P value of less than 0.05 was used as the criterion of statistical significance, and all statistical tests were two-sided. All analyses were performed with SPSS software package .Pearson’s χP>0.05), indicating that the frequency matching was adequate . In addition, more alcohol drinkers among ESCC cases were observed than among controls in these two sets .We observed no statistically significant differences between cases and controls for Jinan case-control set and Huaian case-control set in terms of median age and sex distribution    . Similar df = 1) .MDM4 rs4245739 A>C SNP and ESCC risk  or 0.66  for developing ESCC in Jinan Set or Huaian set, respectively, compared with individual having the rs4245739 AA genotype. In Jinan set, the rs4245739 AC and CC genotypes had a 0.54-fold decreased risk for ESCC compared with the rs4245739 AA genotype . Similarly, logistic regression analyses revealed that individuals with the rs4245739 AC and CC genotypes were also significantly associated with decreased ESCC risk in Huaian set  (P = 0.002)   . In the .6×10−4) . All ORsMDM4 rs4245739 genotypes was further examined by stratifying for age, sex, smoking and alcohol drinking status using the combined data of two Chinese case-control sets (P = 0.023) and subjects aged older than 57 years . No significant gene-age interaction was observed (Pinteraction = 0.796). Compared with the MDM4 rs4245739 AA genotype, a significantly decreased risk of ESCC was associated with AC and CC genotypes only among females , but not among males . There was a marginally significant gene-sex interaction (Pinteraction = 0.080).The risk of ESCC associated with the rol sets . In straMDM4 rs4245739 polymorphism and these risk factors (P = 0.002).There was no significantly decreased risk  for smokers with AC and CC genotypes compared with AA smokers. A multiplicative gene–smoking interaction was also found with Pinteractioin equaling to 0.022. Nondrinkers carrying rs4245739 AC and CC genotypes showed significantly decreased risk to develop ESCC compared with AA carriers who did not drink . However, no association between rs4245739 AC and CC genotypes and ESCC risk was observed in drinkers  (Pinteractioin = 0.032).Because tobacco smoking and alcohol drinking are predisposing factors for ESCC, we then investigated whether a gene–environment interaction existed between the  factors . In nons= 0.799) . An evidMDM4 mRNA and increased MDM4 expression in cancer cells, we studied if there is an allele-specific effect of rs4245739 SNP on MDM4 expression in esophagus tissues. As shown in MDM4 mRNA levels (mean ± SE) than those with AA genotype in normal esophagus tissues , but not in ESCC tissues .Because rs4245739 C-to-A change could destroy RNA::RNA interaction between miR-191 and MDM4 functional SNP and risk of developing ESCC in a case-control design. To the best of our knowledge, this is the first case-control study to investigate the association between the MDM4 rs4245739 polymorphism and ESCC risk. We found significantly decreased ESCC risks among carriers of MDM4 rs4245739 C allele compared with those with A allele in Chinese. In the genotype-phenotype correlation analysis of 29 human ESCC and paired esophagus tissue samples, rs4245739 AC and CC genotypes were associated with a statistically significant decrease of MDM4 mRNA expression. These results are consistent to functional relevance of rs4245739 polymorphism in miR-191-mediaed regulation of MDM4 expression in malignant transformation of human cells In this study, we employed a hypothesis-driven approach to examine the association between a P53 Arg72Pro and MDM2 T309G) have been identified and associated with ESCC risk MDM4 rs4245739 C allele carriers result from the increased tumor suppressor activity of P53.Some functional naturally occurring genetic variants in genes of the P53 tumor suppressor pathway (i.e. MDM4 rs4245739 polymorphism. This could explain, at least in part, the gene-smoking and gene-drinking interaction in the current study.Both tobacco smoking and alcohol are well-known environmental causes of DNA damage MDM4 rs4245739 SNP was associated with a significantly decreased risk of ESCC in Chinese populations. The associations between SNPs and ESCC risk are especially noteworthy in individuals who did not smoke or drink. Additionally, these results may support the hypothesis that genetic variants can interrupt miRNA-mediated gene regulation and this kind of regulatory SNPs might be important modifiers of ESCC risk.In all, our results demonstrated that functional