Aedes aegypti are known to vary with respect to many abiotic and biotic factors including temperature, resource availability, and intraspecific competition. The relative importance of these factors and their interactions are not well established across populations. We performed meta-analysis on a dataset of development rate estimates from 49 studies.Development rates of Ae. aegypti. While diet and density may greatly impact other developmental phenotypes, these results suggest that for development rate these factors should never be considered to the exclusion of temperature. The effect of temperature on development rate is not homogenous or constant. The sources of heterogeneity of the effect of temperature are difficult to analyze due to lack of consistent reporting of larval rearing methods.Meta-analytic results indicated that the environmental factor of temperature is sufficient to explain development rate variability in Ae. aegypti, but its effect is heterogeneous. Ignoring this heterogeneity is problematic for models of vector population and vector-borne disease transmission.Temperature is the most important ecological determinant of development rate in  The effect of temperature on growth has been studied across a wide diversity of organisms -6. Like Aedes aegypti (Linnaeus) is critical to preventing dengue infection [Ae. aegypti, insecticide resistance [Particularly in insects of medical importance, such as mosquitoes that vector human pathogens, estimates of developmental characteristics and models of developmental timing are used to guide vector population control efforts . In partnfection , as thernfection . In Ae. sistance ,28 and csistance  underscosistance  motivatesistance ,32.Few studies have sought to determine importance of other conditions of the developmental environment relative to temperature to explain individual variation in development rate ,33. PlasTo test these hypotheses in diverse environmental conditions, empirical data is needed that considers development rate 1) in response to multiple factors , 2) overAe. aegypti has been well-studied, as it vectors several human pathogens including yellow fever, dengue, and chikungunya, [Ae. aegypti with an aim toward summarizing the impact of multiple environmental conditions on developmental duration, determining the relative importance of these factors, and evaluating their interactions. The conditions evaluated here include temperature, food concentration, food type, larval rearing density, geographic location, and latitude. The linear relationship between development rate and temperature was also evaluated across studies to test the hypothesis that it is a fixed characteristic of the species.ungunya, ,52. We cAe. aegypti development. Of the two forms of Ae. aegypti, Ae. a. formosus, was not included because of known differences in ecology [. The list of databases searched along with keywords and the number of papers included from each source is summarized in supplementary materials (Table S1). The inclusion criteria were as follows. Studies had to report i) the larval rearing temperature, ii) the development time of mosquitoes from hatch to pupation or hatch to emergence in hours or days , iii) the number of replicates, and iv) the number of larvae included for each estimate. In order to accomplish a meta-analysis, datasets must have similar experimental designs [For the literature search and meta-analysis we adhered to PRISMA guidelines. We searched online databases for peer-reviewed research papers in December of 2011 pertaining to  ecology , behavio ecology , and spa ecology  with lim ecology . The lis designs , and we We used two meta-analytic approaches for these data. In our first approach we evaluated estimates of development time from hatch to pupation and development time from hatch to emergence using a mixed linear regression model  “nlme”  implemenAe. aegypti are well approximated by a linear model within the temperature range from 14 - 31°C [In our second approach, we focused analysis on a temperature range for which development rate (1/development time) can be well approximated with a linear model. Development rates in 4 - 31°C . The linB0 represents the developmental zero and B1 is a constant for the cumulative effective of temperature, generally reported as K[B1 and B0 were each used as effect measures, and were weighted by the number of replicates per experiment in a study. We tested the hypotheses that cumulative effect of temperature (K) and developmental zero (t) are constant properties of a mosquito strain using a test of total heterogeneity, QT, with Hedge’s estimator, a standardized difference method for comparing effect measures [E[where y = 1/development rate, and y is regressed on temperature, x. The parameter rted as K,109. Whemeasures ,110. Nexasures [E,110. ForAedes aegypti, temperature, diet, larval rearing density, and development rate, we found 27,559 articles, from which 48 journal publications and one book chapter fit the inclusion criteria  and hatch to emergence . A linear mixed effects model was used to determine what other environmental factors might explain the heterogeneity in this relationship. Additional factors considered were initial larval rearing density, diet level (mg/larva/day), strain origin, latitude, and publication author. The model including only temperature as a fixed factor and the random factor of publication author best explained the heterogeneity in slope estimates for both the pupation group and emergence group. Once publication was included in the model, the test of residual heterogeneity was no longer significant for hatch to pupation  or hatch to emergence . Similarly, the developmental zero was significantly heterogeneous for both the hatch to pupation development rate  and hatch to emergence . Once temperature had been considered, the residual heterogeneity in the developmental zero was explained by publication author such that the test for residual heterogeneity was no longer significant .The linear association between development rate and temperature had significant heterogeneity for both hatch to pupation . Of the 49 studies, almost all reported information on diet composition. However, few used the same diet preparations, and this prevented this factor from being included in meta-analysis. Some diets were created from detritus of the larval habitat in order to mimic natural conditions ,61,137 oprograms ,58,69.Ae. aegypti populations [Ae. aegpyti that could explain the heterogeneity of responses characterized here. Further, in natural conditions other ecological factors not considered here such as interspecific competition, such as between Ae. aegypti and Ae. albopictus[, and predation [Other factors not considered in this analysis may also impact the effect of temperature, and perhaps contribute to heterogeneity. Examples include genetic variation, microbial symbiotic partners, and maternal effects. Population differences in larval survival and body size in response to different temperatures have been demonstrated in other insects  but suchulations . Richardulations  suggestelbopictus,137, andredation  may impaRecent work compares life-history traits such as body size and fecundity across multiple environmental conditions ,146. MorAe. aegpyti[Ae. aegypti but that the effect is heterogeneous. Ignoring the heterogeneity in response to temperature may be problematic for using development rate estimates to model vector populations and predicting the impact of temperature on vector-borne disease transmission.Beyond utility for vector population control, development rate estimates may be useful for modeling and understanding disease transmission. There is evidence that larval environment impacts adult dispersion of . aegpyti as well . aegpyti. Depinay. aegpyti have demAll authors have read and understood the BMC Ecology policy on declaration of competing interests and declare: no financial relationships with any organizations that might have an interest in the submitted work; no other relationships or activities that could appear to have influenced the submitted work; no patents relating to the content of the manuscript; no financial or non-financial competing interests.JC conceived the study, acquired data through a literature search, analyzed and interpreted data, and drafted manuscript. MQB acquired data through literature search, interpreted data, and revised manuscript critically for important intellectual content. Both authors have read and approved the final manuscript.Aedes aegypti development rate estimates under various environmental conditions including temperature, diet and intraspecific rearing density. Databases are ordered based on specificity to mosquito literature from broad to specific. Table S2. Full bibliography for the 65 studies included in the factors influencing development rate and survival of Aedes aegypti. Table S3. Linear regression parameter estimates for studies that experimentally examined the relationship between development rate and temperature for the stages from first instar to adult emergence. Table S4. Linear regression parameter estimates for studies that experimentally examined the relationship between the development rate and temperature for the life stages from hatch to pupation.Online database searched in December 2011 for research papers pertaining to Click here for fileB1- the slope of the regression of temperature and development rate from hatch to emergence. (A) Forest plot of best model with random effect of publication author. (B) Funnel plot corresponding to plot (A). The weight of the study is indicated by the size of the square and the diamond indicates the overall effect estimate from the random effects model. Squares represent effect estimates of individual studies. Square size represents the weight given to the study in the meta-analysis, and the horizontal lines represent 95% confidence intervals. Estimated values and confidence intervals are written to the right of the plot. In the funnel plots, dots represent the residuals of the publication authors corresponding with the best model and their associated standard error. When the residuals fit within the light cone, it implies that heterogeneity in the main effect is successfully accounted for by the model.Meta-analysis of the effect of temperature, i.e. Click here for fileB1- the slope of the regression of temperature and development rate from hatch to pupation. (A) Forest plot of best model with random effect of publication author. (B) Funnel plot corresponding to plot (A). See Figure S1 caption.Meta-analysis of the effect of temperature, i.e. Click here for file


DNA repair genes  may affect the capacity of encoded DNA repair enzymes to effectively remove DNA adducts or lesions, which may result in enhanced cancer risk. The association between XPD gene polymorphisms and the susceptibility of prostate cancer (PCa) was inconsistent in previous studies.XPD Gln751Lys SNP (single nucleotide polymorphism) and 2555 cases and 3182 controls for Asn312Asp SNP was performed to address this association. Meanwhile, odds ratio (OR) and 95% confidence intervals (CIs) were used to evaluate this relationship. Statistical analysis was performed with STATA10.0. No significant association was found between XPD Gln751Lys SNP and PCa risk. On the other hand, in subgroup analysis based on ethnicity, associations were observed in Asian  and African  populations for Asn312Asp SNP. Moreover, similar associations were detected in hospital-based controls studies; the frequency of Asn/Asn genotype in early stage of PCa men was poorly higher than those in advanced stage of PCa men .A meta-analysis based on 9 independent case-control studies involving 3165 PCa patients and 3539 healthy controls for XPD Asn312Asp SNP not the Gln751Lys SNP, might poorly increase PCa risk in Asians and Africans, moreover, this SNPs may associate with the tumor stage of PCa. Further studies based on larger sample size and gene-environment interactions should be conducted to determine the role of XPD gene polymorphisms in PCa risk.Our investigations demonstrate that  Prostate cancer (PCa) is the most common male non-dermatological cancer in Europe and the USA, and the sixth leading cause of cancer related-deaths, accounting for 14%  of total new diagnosed cancer cases and 6%  of whole cancer deaths in males in 2008 Various DNA alterations can be caused by exposure to environmental and endogenous carcinogens, including ultraviolet (UV) light, cigarette smoke, dietary factors, reactive oxygen species, and carcinogens. Most of these alterations, if not repaired, can result in genetic instability, mutagenesis and cell death. Because DNA repair pathways (DRP) play a critical role in maintaining the genomic integrity in general and specialized functions of cell as well as in the prevention of carcinogenesis, therefore the defection of those genes in DRP can lead to highter susceptibility to multiple cancers There are a number of DRP, each responsible for repairing a different type of DNA damage. Base excision repair (BER) removes simple base modifications, including single-strand breaks, oxidative DNA damage, and alkylation and nonbulky adducts The xeroderma pigmentosum group D (XPD) gene encoding for NER protein is located on chromosome 19q 13.3. It comprises 23 exons and spans about 54,000 base pairs G→A substitution causing exon 10 codon 312 Asp to be exchanged for Asn  and an A→C substitution causing exon 23 codon 751 Lys to be substituted for Gln  Two common non-synonymous single-nucleotide polymorphisms (SNP) in the coding region of the XPD gene have been identified: a Several epidemiologic studies have examined associations between SNPs in DNA repair genes, mostly non-synonymous SNPs in XPD gene with potential functional significance and risk of PCa Asn312Asp and Gln751Lys) and PCa risk up to March 20, 2012. The medical subject headings and key words used for search were ‘XPD or ERCC2 or xeroderma pigmentosum group D or excision repair cross-complementing rodent repair deficiency Group 2’, and ‘prostate cancer or tumor’, and ‘polymorphism or variant’. The electronic searching was supplemented by checking reference lists from the identified articles and reviews for additional original reports. All of studies must meet the following criteria: (1) study was designed using the methodology of a case-control study; (2) the association between XPD Asn312Asp and/or Gln751Lys polymorphisms and PCa risk was explored; (3) cases with carcinomas were diagnosed by histopathology. The major exclusion criteria were: (1) duplicate data, (2) abstract, comment, review and editorial, and (3) no sufficient data were reported. All studies were published in English language.We searched the Pubmed and Embase databases for all articles on the association between XPD two polymorphisms  extracted all data independently, complied with the selection criteria, and reached a consensus on all items. In case of disagreement, a third author (Zhang) assessed the articles. The following items were collected: first author's last name, year of publication, country of origin, ethnicity, source of control  and Hardy–Weinberg equilibrium (HWE) of control, total number and genotype distributions in cases/controls and genotyping method. For studies including subjects of different ethnicities, data were extracted separately and categorized as Caucasians, Asians and Africans. Agalliu et al. Gln/Gln vs. Gln/Lys for 751 SNP, Asn/Asn vs. Asn/Asp for 312 SNP), homozygote comparison , dominant and recessive models  and allelic comparison , respectively. The heterogeneity among the studies was checked by using the chisquare based Q statistic and considered statistically significant at P<0.05 P>0.05, the pooled OR of each study was calculated by using the fixed-effects model ; otherwise, the random-effects model (the DerSimonian and Laird method) was used Z-test, and P<0.05 was considered statistically significant. The departure of frequencies of XPD polymorphisms from expectation under HWE was assessed by the chi-square test in controls and a P<0.05 was considered as significant disequilibrium. Publication bias was diagnosed with Egger's linear regression method and funnel plot. The P-value less than 0.05 in Egger's linear regression indicated the presence of potential publication bias The strength of the association between the XPD two polymorphisms and PCa risk was measured by odds ratios (ORs) with 95% confidence intervals (CIs). Pooled ORs were obtained from combination of single studies by heterozygote comparison  for Asn312Asp polymorphism to evaluate the association with risk for PCa, respectively (Table S1). The distribution of genotypes among controls was consistent with HWE in all studies except two A total of 29 articles were achieved by literature search from the Pubmed and Embase, using different combinations of key terms. As shown in Asn312Asp/Gln751Lys) polymorphisms and PCa susceptibility for all genetic models (Table S2). After excluding two studies not agreement with HWE, the overall association was not changed. We did not find any significant results when we stratified the studies by ethnicity and source of the controls between Gln751Lys polymorphism and PCa risk.The results of the overall meta-analysis did not suggest any associations between two XPD  and Africans . Similar relationships were reported in HB source of the controls subgroup (Table S2).For Asn/Asp+Asp/Asp genotypes, PCa cases with Asn/Asn genotype showed poorly higher percentage value in early stage group, but not in advanced stage group .Interestingly, compared to Table S3).We use sensitivity analysis to determine whether modification of the inclusion criteria of the meta-analysis affected the final results. Finally, no other single study influenced the summary OR qualitatively as indicated by sensitivity analysis (data not show). The Egger's test was performed to access the publication bias of literatures, which was used to provide statistical evidence of funnel plot symmetry. Ultimately, the results did not suggest any evidence of publication bias (Asn312Asp/Gln751Lys) and PCa risk by using meta method to get a powerful conclusion. The potential role of XPD two polymorphisms as determinant of PCa risk was investigated in a sample of 6752 subjects (3165 cases and 3539 controls for Gln751Lys and 2555 cases and 3182 controls for Asn312Asp) from nine published case-control studies.The identification of SNPs that affect gene function or expression and contribute to PCa susceptibility is important to help predict individual and population risk and understand the pathogenesis of PCa. Rybicki et al. first found XPD two common polymorphisms to be associated with the risk of PCa in a Caucasian population in 2004 Asn312Asp or Gln751Lys polymorphism. Similarly, Rybicki et al. 312Asn/Asn or 751Gln/Gln, respectively). Lavender et al. 312 Asp/Asp genotype, however, there were no significant differences in the allele frequencies between cases and controls for Gln751Lys polymorphism. Our study showed similar observation with Lavender et al. Little is know for sure about the underlying mechanism for this association. The XPD gene has been mapped in chromosome 19q13.3. It spans over 20 kb, contains 23 exons and encodes the 761-amino acid protein. The XPD protein, on one hand, possesses both single-strand DNA-dependant ATPase and 5′-3′ DNA helicase activities, which is essential for NER pathway and transcription; on the other hand, is absolutely necessary for efficient DNA repair capacity, which is essential for the maintenance of genetic stability and associated with cancer susceptibility when incompetent XPD Asn312Asp not Gln751Lys polymorphism and PCa among Asian and African populations. There must be some factors that would contribute to this discrepancy. First, multiple genes and environmental factors may lead to PCa formation. Second, ethnicity may be related to PCa incidence. For example, PCa is the most common non-cutaneous malignancy and the second leading cause of cancer mortality in Western men. In contrast to the trends of western countries, incidence and mortality rates are rising in several Asian and central/eastern-European countries, such as Japan, China and Poland, suggesting an increasingly westernized lifestyle in these regions In present study, no significant association was found under any genetic model in the overall analysis. However, in the stratified analysis by ethnicity, significant increased associations were detected between 312Asn/Asn genotype in early stage cases was more higher than in advanced stage cases, in addition, Asn/Asn genotype was associated with increased PCa risk, thus, we predicted 312Asn/Asn genotype could poorly help us to detect PCa and evaluate its prognosis. No association was detected between clinical characteristics of PCa and Gln751Lys SNP.We all know that the Gleason score grading system has withstood the test of time as a predictor of PCa outcome. Additionally, the tumor stage of PCa is central to management because it contributes both to predicting prognosis and planning treatment. In the current study, the frequency of PCa cases carried Gln751Lys and also two studies about tumor stage about Asn312Asp, the number of which was small, further studies should increase these relations to enlarge the subsequent meta-analysis. Fourth, we classified the tumor stage into ‘early stage’ [localized stage or none bone metastasis (−)] and ‘advanced stage’ [regional/distant stage or bone metastasis (+)], however, the criteria of patient's selection in these two studies were not quite concordant. In spite of these, our meta-analysis also had three advantages. First, substantial number of cases and controls were pooled from different studies, which significantly increased statistical power of the analysis. Second, the quality of case–control studies included in the current meta-analysis was satisfactory based on our selection criteria. Third, the control groups were all healthy people.Although, we have put considerable efforts and resources into testing possible association between XPD polymorphisms and PCa risk, there are still some limitations inherited from the published studies. First, although we collected all the eligible studies, the sample size of the included studies was not large enough, which could increase the likehood of type I and type II errors. Therefore, there was a lack of statistical power to better evaluate the association between XPD polymorphisms and PCa risk, especially in subgroup analysis by ethnicity. Second, gene-gene and gene-environment interactions were not analyzed. It is possible that specific environmental and lifestyle factors may alter those associations between gene polymorphisms and PCa. Therefore, it is necessary to evaluate the roles of some special environment factors and lifestyles such as diet, alcohol consumption and smoking status and cancer family history. Third, two studies refered to Gleason score for XPD Asn312Asp not Gln751Lys polymorphism may contribute to genetic susceptibility for increased PCa risk in Africans and Asians. Furthermore, XPD Asn312Asp polymorphism was associated with PCa prognosis and/or outcome. Large population studies are needed to be performed to clarify possible roles of XPD polymorphisms in the etiology and clinical characteristics of PCa.In conclusion, present analysis suggested that Table S1Study characteristics from published studies on the relationship between XPD gene two polymorphisms and PCa.(DOC)Click here for additional data file.Table S2Total and stratified analysis of XPD gene two polymorphisms on PCa.(DOC)Click here for additional data file.Table S3Publication bias tests (Egger's funnel plot for publication bias test) for XPD gene two polymorphisms.(DOC)Click here for additional data file.


KIF6 Trp719Arg to both the risk of CHD and the efficacy of statin therapy in CHD patients.The goal of our study is to assess the contribution of P<0.001, HR = 1.27, 95% CI = 1.15–1.41). However, another meta-analysis of 7 case-control studies among 65,200 individuals fails to find a significant relationship between Trp719Arg and the risk of CHD . This suggests that the contribution of Trp719Arg to CHD varies in different ethnic groups. Additional meta-analysis also shows that statin therapy only benefit the vascular patients carry 719Arg allele  = 0.60, 95% CI = 0.54–0.67). To examine the role of this genetic variant in CHD risk in Han Chinese, we have conducted a case-control study with 289 CHD cases, 193 non-CHD controls, and 329 unrelated healthy volunteers as healthy controls. On post hoc analysis, significant allele frequency difference of 719Arg is observed between female CHD cases and female total controls under the dominant model  = 1.979, 95% confidence interval (CI) = 1.023–3.828). Similar trends are observed for post hoc analysis between female CHD cases and female healthy controls . Non-genetic CHD risk factors are not controlled in these analyses.Meta-analysis of 8 prospective studies among 77,400 Caucasians provides evidence that 719Arg increases the risk of CHD (KIF6 gene in the risk of CHD in Caucasians. The meta-analysis also suggests the role of this variant in statin therapeutic response in vascular diseases. Our case-control study suggests that Trp719Arg of KIF6 gene is associated with CHD in female Han Chinese through a post hoc analysis.Our meta-analysis demonstrates the role of Trp719Arg of  Severe coronary artery disease (CAD), also called coronary heart disease (CHD), is characterized by occlusive epicardial coronary artery stenosis. CHD complications such as myocardial infarction (MI) are the leading causes of death in the United States KIF6) gene encodes an intracellular motor protein transporting cellular cargos along microtubules in an ATP dependent process. KIF6 gene is expressed in many tissues and cell types including coronary arteries and vascular cells KIF6 such as Trp719Arg (rs20455) have been shown to be associated with the risk of CHD Kinesin like protein 6 , Wanfang Chinese Periodical Database and Web of Science. The search keywords apply the MeSH  terms that include “coronary heart disease” or “coronary artery disease” or “myocardial infarction” combined with “KIF6” or “kinesin like protein 6” or “rs20455” or “719Arg”, “polymorphism”, “genetic association” and/or “statin” Data extraction is carried out by at least two reviewers (PP and LMX) on a standard protocol, and the consensus data are established by discussion. In the meta-analyses, the following data collection is included: name of the first author, publication year, country, ethnic population, study stage, numbers of individual in the case and the control groups and prospective studies, OR, RR, HR and 95% CI. The meta-analyses are performed by Stata software  A total of 289 CHD patients and 193 non-CHD patients are collected between May of 2008 and November of 2011 from the Lihuili Hospital in Ningbo city of Zhejiang province, China. Patients are differentiated into case and control group by standardized coronary angiography according to the Seldinger's method 5′-ACGTTGGATGTTCTCCAGACATCTGACTCC-3′ and the antisense primer is 5′-ACGTTGGATGCCGGTGAGTTCTCACCTTAC-3′. PCR conditions include an initial denaturation stage 94°C for 15 sec, followed by 45 cycles at 94°C for 20 sec, 56°C for 30 sec, primer extension at 72°C for 1 min, and a final extension for 3 minutes at 72°C. Primer Extension for genotyping is performed on the SEQUENOM® Mass-ARRAY iPLEX® platform according to the manufacturer's instructions Human genomic DNA is isolated from peripheral blood using a conventional phenol/chloroform extraction method, and is quantified using the PicoGreen® double strand (dsDNA) DNA Quantification Kit . Amplification is performed on the ABI Geneamp® PCR System 9700 Dual 384-Well Sample Block Module  for the Polymerase Chain Reaction (PCR). The sense primer is P<0.05 is considered to be significant.Genotype distribution of different sample groups is tested for departure from Hardy-Weinberg equilibrium (HWE) by the Arlequin program (version 3.5) P<0.001, the overall HR = 1.27, 95% CI = 1.15–1.41) and a high heterogeneity among the 8 prospective studies . In contrast, a low heterogeneity is observed in the meta-analysis of the six case-control studies and our study . The latter meta-analysis has not found a significant association between 719Arg and the risk of CHD  despite of the large sample size . This observation along with the mixed ethnicity in this meta-analysis, we speculates that the contribution of Trp719Arg to the risk of CHD varies in different ethnic groups. No publication bias is observed among the involved studies in the two meta-analyses  is found among the eight studies. Random effects analysis model is used for the meta-analysis. No publication bias is observed among the involved studies by the funnel plot . Interestingly, an association test in females shows significant different distribution of 719Arg-containing genotypes between CHD cases and healthy controls under the dominant model . This finding remains true between CHD cases and total controls under the dominant model for females . Note that post hoc analysis of our results generates the hypothesis that 719Arg tends to have a dominant effect on the risk of CHD (OR = 2.012) only in female Eastern Han Chinese . This female-specific finding agrees with the observations in a total of 25,283 Caucasian women enrolled in the WHS P<0.001, HR = 1.27, 95% CI = 1.15–1.41). This result agrees with a previous meta-analysis that has found a 20% increase in the risk of CHD for the 719Arg carriers Several lines of evidence have shown that 719Arg is likely to increase the risk of CHD We also notice that there is an ethnic difference in the frequency of 719Arg allele. In our healthy controls, it is 0.483 that is similar to 0.570 in HapMap-CHB, 0.477 in HapMap-JPT, and 0.51 in the Indian population No significant association is found between 719Arg and the risk of CHD in the meta-analysis of 7 case-control studies among 65,200 individuals. The recruited participants in the meta-analysis are from several different ethnic populations including Europeans or European descendents, African descendents in America, East Asians, and South Asians. Among these case-control studies, a large one with a total of 17,000 cases and 39,369 controls failed to replicate the association between Trp719Arg and the risk of clinical CHD in multiple ethnic populations P = 0.007, HR = 0.61, 95% CI = 0.43–0.87) and non-carriers  of 719Arg An allele-specific model of 719Arg is observed in the statin therapy of coronary events. Significantly reduced coronary events and other major vascular events have been observed in 719Arg carriers but not in non-carriers KIF6 gene in CHD risk and coronary event reduction from statin therapy has yet to be clearly elucidated Mechanistically, the structure of KIF6 protein consists of a conserved motor domain and a non-conserved tail domain. The conserved motor domain can propel the kinesin along microtubules in an ATP-dependent manner. The non-conserved tail domain binds to its cargoes such as membrane organelles, protein complexes, and mRNAs P = 0.041). This phenomenon may be due to the improperly pair-wised design, the small population size or that the female CHD patients do not obey HWE. Therefore, we need to take caution with the significant association results in females under the dominant model. Thirdly, the information of statin usage is not available in our samples. Our study might underestimate the risk of this genetic variation for a potential bias by the statin therapy. Moreover, CHD is strongly correlated with smoking status, LDL-C/HDL-C, history of hypertension or diabetes, and body mass index. Our genetic testing has not been adjusted with those risk factors. Fourthly, gender has been shown to be an important modifier of cardiovascular disease risk PDGFD gene variation (rs974819) and CHD risk. In the present meta-analyses, we are unable to make a gender adjustment or stratification for all the involved studies. Finally, our study isn't designed to test whether KIF6 variants are associated with the statin therapy outcome.There are several limitations in our case-control study focused on Han Chinese. Firstly, sample size in our study is comparatively small and it has only 20.6% power to detect the association of Trp719Arg with CHD at a significant level of 0.05. In addition, only non-fatal CHD cases are recruited in the present study. The 719Arg allele is hypothesized to increase the risk of incident fatal CHD more than the risk of incident nonfatal CHD KIF6 719Arg allele. Despite failing to find a significant relationship between Trp719Arg and CHD in Eastern Han Chinese for the study population as a whole, post hoc analysis reveals a female-specific association. This gender-specific finding should be investigated future studies.In conclusion, our meta-analyses over 143,000 individuals have shown that 719Arg is a risk factor of CHD in Caucasians but its effects on CHD may vary in other ethnic populations. Meta-analysis also indicated that statin therapy may selectively benefit patients with Table S1Association of Trp719Arg with CHD in the dominant model.(DOCX)Click here for additional data file.Table S2Association of Trp719Arg with CHD in the recessive model.(DOCX)Click here for additional data file.Table S3Association of Trp719Arg with gender risk in the recessive model.(DOCX)Click here for additional data file.Flow Diagram S1PRISMA flow diagram.(DOC)Click here for additional data file.Checklist S1PRISMA checklist.(DOC)Click here for additional data file.


RNLS), a recently discovered enzyme with monoamine oxidase activity, is implicated in the degradation of catecholamines. Recent studies delineate a possible role of this enzyme in blood pressure (BP) maintenance and cardiac protection and two single nucleotide polymorphisms, RNLS rs2576178 A > G and rs2296545 C > G have been associated with hypertension. The latter SNP leads to a non synonymous Asp to Glu substitution deleting a flavin adenine dinucleotide (FAD) binding site with possible impaired functionality. We tested the hypothesis that these polymorphisms could affect BP levels, hypertension prevalence, and risk of incident cardiovascular events in middle-aged Swedes.Renalase . The incidence of cardiovascular events  was monitored for an average of approximately 15 years of follow-up.Both before and after adjustment for sex, age and BMI the polymorphisms did not show any effect on BP level and hypertension prevalence. Before and after adjustment for major cardiovascular risk factors, the hazard ratio for cardiac and cerebrovascular events was not significantly different in carriers of different genotypes. A significant interaction was found between the rs2296545 C > G and age with respect to BP/hypertension.RNLS polymorphisms in determining BP level and incident events at population level. The positive interaction with age suggest that the effect of the rs2296545 C > G polymorphism, if any, could vary between different ages.Our data do not support a major role for these  RNLS) is a recently discovered enzyme with monoamine oxidase activity implicated in the degradation of catecholamines and probably other currently unknown substrates GGTAAATTCTTCTTCCCACTTTCAAVIC/FAM-TGGCGTTTAGACAACCCACCTGAGTC[C/G]TCAGCCTTGTCCCACACAGCAAGGTt-test. Multiple linear and logistic regression analyses were used in the multivariate models with either BP traits or hypertension status as dependent variables and genotype, age, sex, BMI, and the interaction variables  as independent variables.Continuous variables are presented as the mean ± SD. All data, except for the power analysis, were analyzed with SPSS statistical software . Power calculation was performed using the Power and Sample Size calculator version 2.1.31 . Frequency differences and deviation from Hardy- Weinberg equilibrium were analyzed by chi-square test. Significance of differences in continuous variables was tested by analysis of variance followed by Tukey’s test and P values less than 0.05 were considered statistically significant.Kaplan-Meier curves and log-rank tests compared cumulative incidence of strokes, coronary events, acute heart failure and atrial fibrillation/flutter episodes in carriers of different genotypes. Age-, sex- and traditional risk factor  adjusted Cox proportional-hazard models were used to study the relationships between the polymorphisms and time (in years) to first cardiovascular events. The fit of the proportional hazards model was confirmed by plotting the cardiovascular incidence rates over time. Hazard ratios (HR) and 95% confidence interval (CI) were calculated. For variables with skewed distributions, log-normalized values were used in the analysis. All tests were two-sided and RNLS rs2576178 A > G and 97.5%  for the rs2296545 C > G. We found respectively for the rs2576178, 53.6% AA homozygotes, 38.5% AG-heterozygotes and 7.9% GG-homozygotes and for the rs2296545, 29.7% CC-homozygotes, 49.4% CG-heterozygotes and 21.0% GG-homozygotes.The clinical characteristics of individuals included in the study in the MDC-CVA are summarized in Table P = 0.05 for RNLS rs2576178 A > G and predicted heterozygosity 0.494, observed heterozygosity 0.496; P = 0.70 for rs2296545 C > G).Genotype distributions did not deviate from Hardy–Weinberg equilibrium for both SNPs  for hypertension greater than 1.31/1.20 according to an autosomal recessive genetic model (AA&AG vs. GG and CC&CG vs. GG), greater than 1.17/1.19 according to a dominant (AA vs. AG&GG and CC vs. CG&GG) genetic model and greater than 1.13/1.12 according to an additive genetic model.Regarding the RNLS rs2576178 A > G SNP, if an autosomal dominant/recessive/additive genetic model is assumed, the study has 80% power to detect a difference of respectively >1.41/>2.61/>1.12 mmHg in systolic BP and >0.70/>1.29/>0.55 mmHg in diastolic BP between subjects carrying different genotypes. Analyzing the RNLS rs2296545 C > G SNP, if an autosomal dominant/recessive/additive genetic model is assumed, the study has 80% power to detect a difference of respectively >1.55/>1.74/>1.01 mmHg in systolic BP and >0.77/>0.87/>0.50 mmHg in diastolic BP between subjects carrying different genotypes.Regarding continuous BP variables, analyzing the Crude BP data and hypertension prevalence according to genotype at MDC-CVA are presented in Table RNLS rs2576178 A > G genotype  and rs2296545 C > G genotype .Analyzing hypertension prevalence as a dichotomous trait we did not find any difference in carriers of different P > 0.05 for all). The same analysis were repeated after exclusion of subjects with previous cardiovascular events (n = 117), diabetes mellitus (n = 443) and CKD (n = 38) and after adjusting for other metabolic covariates but the results did not change significantly  and individuals with BP below 130/85 mmHg  showed no significant difference in genotype frequencies , diastolic BP  and hypertension  in the lowest quartile of age . Whereas a trend toward higher systolic BP , diastolic BP  and hypertension  was observed in the highest quartile of age . Hypothesizing that this interaction could be due to a decrease in renal function with aging we performed an exploratory analysis, within a subsample where eGFR cystatin was available . No association between the rs2296545 C > G polymorphism and hypertension-related traits was detectable  with respect to our study (0.27) [RNLS Asp37Glu were referred to echocardiographic measurements of hypertrophy and inducible ischemia that were not evaluated in our study [Our study, at variance with previous reports, found no association with hypertension related traits in a well-powered sample of middle-aged men and women participating in the MDC-CVA. There are different possible explanations for this discrepancy. Previous reports refer to population collected with different design, selection criteria, clinical characteristics and genetic background. Association of the ur study ,22. AlsoThe positive interaction we found between the rs2296545 (Asp37Glu) and age with respect to hypertension, suggests that the Glu37 variant should be protective at lower ages and deleterious at advancing ages. This finding is difficult to explain and despite having found several times interaction with sex in the same population sample, this is first time we detected such kind of interaction -25. We cWhereas further ad hoc study try to clarify this issue, we invite to consider carefully the age of investigated people when this particular polymorphism is tested for association with hypertension.During a follow-up of approximately 15 years also “hard” end points, such as myocardial infarction, strokes, atrial arrhythmic events and heart failure were not associated with these polymorphisms in our population. Even if we found a borderline significant results suggesting as a protective role of these SNPs about coronary events and atrial fibrillation episodes, our findings would go exactly in the opposite direction respect to what could be expected on the basis of previous results about hypertension in Japanese people and cardiac hypertrophy in CAD patients with the putatively “deleterious” minor alleles conferring a protection respect to these cardiovascular events. Indeed, the accordance we have about results on BP and cardiovascular end-points suggest a negligible effect of this polymorphism, at least in our population.RNLS rs2296545 which results in an Asp to Glu substitution at codon 37, which is located near a deduced FAD-binding site of the renalase protein.We focused our attention especially on the RNLS rs2576178 there are no functional data in vitro but the polymorphism is in the 5’ flanking region of the gene and could be involved in transcriptional regulation. However, we cannot exclude that other SNPs in the same gene in variable grade of linkage disequilibrium with our SNPs could be implicated in BP/hypertension or other cardiovascular related outcomes.Since renalase is critically dependent on FAD for oxidase activity, the polymorphism could be functional. For the Strengths of our study are the large sample size and the population-based-design with prospective assessment of cardiovascular end-points; whereas limitations refers especially to the measurement of BP at a single-time point. Indeed, our results are not generalizable to populations with different genetic background.RNLS SNPs, hypertension and cardiovascular events suggesting that at population level these polymorphisms are of negligible importance, at least in Caucasian.In a Swedish urban-based cohort, including more than 5,000 subjects, we found no evidence of association between two common RNLS on hypertension and BP related phenotypes are linked to selection criteria or different genetic background of the analyzed populations. About other cardiologic outcomes, it will be important to test if at risk population, such as people affected by CAD or with impaired renal function could be more prone to the putative deleterious effect of RNLS polymorphisms. Moreover, a possible effect of age interacting with the Asp37Glu polymorphism should be considered when selecting newer populations where to study this gene.Further studies are needed to elucidate if conflicting results respect to previous studies about the effect of The authors declare that they have no competing interests.CF participated in the study design, carried out the molecular genetic studies, participated in the sequence alignment, performed statistical analyses and drafted the manuscript. MM carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. ED carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. MS carried out the molecular genetic studies and participated in the sequence alignment. PA performed statistical analyses and helped to draft the manuscript. GE, BH, GCG, PM and OM participated in study 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/1471-2350/13/57/prepubTable S1. Association between the RNLS polymorphisms and BP according to different genetic model after excluding subjects with previous CV events, diabetes mellitus and CKD (n=4209). Table S2. Association between the RNLS polymorphisms and BP according to different genetic model after further adjustment for glucose, triglycerides, total cholesterol, high density lipoprotein cholesterol, serum cystatin C, smoking and drinking status (n=4209). Table S3. Beta coefficient and SE for interaction terms (either sex or age) with RNLS rs2576178 and rs2296545 polymorphisms and BP-related traits according to different mode of inheritance. Table S4. Association between the RNLS rs2296545 polymorphism and BP according to different genetic model in subjects with impaired kidney function.Click here for file


A total of 879 CAD patients and 628 controls from eight separate studies were involved. The pooled odds ratio (OR) for the distribution of the 4G allele frequency of PAI-1 4G/5G gene and its corresponding 95% confidence interval (CI) was assessed by the random effect model.The present meta-analysis was performed to investigate the association between PAI-1 4G/5G gene polymorphism and CAD in the Chinese Han population was significant under an allelic genetic model . The heterogeneity test was also significant (P<0.0001). Meta-regression was performed to explore the heterogeneity source. Among the confounding factors, the heterogeneity could be explained by the publication year (P = 0.017), study region (P = 0.014), control group sample size (P = 0.011), total sample size (P = 0.011), and ratio of the case to the control group sample size (RR) (P = 0.019). In a stratified analysis by the total sample size, significantly increased risk was only detected in subgroup 2 under an allelic genetic model .The distribution of the 4 G allele frequency was 0.61 for the CAD group and 0.51 for the control group. The association between PAI-1 4G/5G gene might predispose to CAD.In the Chinese Han population, PAI-1 4G/5G gene polymorphism was implied to be associated with increased CAD risk. Carriers of the 4G allele of the  The plasminogen activator inhibitor-1 (PAI-1), a key regulating factor, determines endogenous fibrinolysis activity. The elevated plasma concentration or activity of PAI-1 is one of the relevant signs of atherosclerosis and thrombus disease PAI-1 gene, located in 7q21.3–22, spans 12.3 kb and contains 9 exons and 8 introns. The polymorphism of the 4G/5G gene is located in the PAI-1 gene promoter region. The 5th guanine (G base) is inserted or deleted in the 4 G sequence in the –675th base of the transcription initial point upstream. The PAI-1 gene has three genotypes, namely, 4G4G, 4G5G, and 5G5G. 4G4G allele carriers always have higher plasma PAI-1 activity than 4G5G and 5G5G carriers The PAI-1 (–675) 4G variant yields a per-allele relative risk of 1.06 (1.02 to 1.10) for coronary disease. However, their study had an indication of publication bias PAI-1 4G/5G gene polymorphism does not affect the severity of CAD in an Indian population In 2006, Ye et al. performed a meta-analysis on seven hemostatic gene polymorphisms in coronary disease in Caucasians and African-Americans. They reported that the PAI-1 4G/5G gene polymorphism and CAD have been extensively performed in China, but the results are still disputable. In 2001, Dai Y et al. reported that PAI-1 4G/5G gene polymorphism is not associated with susceptibility for CAD PAI-1 4G/5G gene polymorphism and CAD in the Chinese Han population .The electronic databases PubMed, Embase, Web of Science, China Biological Medicine Database, and China National Knowledge Infrastructure were searched using the MeSH terms “coronary artery disease” or “coronary heart disease,” “polymorphism,” “PAI-1 4G/5G gene polymorphism and CAD in a Chinese Han population; b) diagnosis of CAD in accordance with the examination results of coronary arteriography (>50% stenosis of at least one major vessel), as well as clinical symptoms combined with electrocardiogram, echocardiography, treadmill exercise test, and myocardial perfusion imaging in emission computed tomography (ECT); c) controls proven to have no CAD either by coronary arteriography or by medical history, blood test, physical examination, electrocardiogram, echocardiography, treadmill exercise test, and ECT; and d) no other cardiovascular risk factors such as hypertension, hyperlipidemia, and diabetes mellitus .The selected studies had to be in accordance with the following major criteria: a) evaluation of The data were obtained according to a standard protocol. Studies that were repeated, did not meet the inclusion criteria, or provided little data were excluded. If the same data appeared in different works, the result was only used once. The collected data comprised the name of the first author, publication year, region, number of genotypes, genotyping, study design, matching criteria, and total number of cases and controls.PAI-1 4G/5G gene polymorphism and CAD was compared using the odds ratio (OR) corresponding to a 95% confidence interval (CI). The presence of inter-study heterogeneity was calculated by the Chi-square-based Q-test, and significance was set at the P<0.05 level I2 was calculated to assess the variation caused by the heterogeneity. If heterogeneity was observed among the studies, the random-effects model was used to estimate the pooled OR (the DerSimonian and Laird method) P<0.05.The association between P<0.05. The potential publication bias was estimated by the funnel plot. Egger’s linear regression test on the natural logarithm scale of the OR was used to assess the funnel plot asymmetry; the significance was set at the P<0.05 level Fisher’s exact test was used to assess the Hardy–Weinberg equilibrium (HWE) with the significance set at PAI-1 4G/5G gene polymorphism. The data were collected from 879 CAD patients and 628 controls   .P<0.0001). Meta-regression was performed to explore the heterogeneity source. Among the confounding factors, the heterogeneity could be explained by the publication year (P = 0.017), study region (P = 0.014), control group sample size (P = 0.011), total sample size (P = 0.011), and RR (P = 0.019). In a stratified analysis by the total sample size, significantly increased CAD risk was detected in subgroup 2 under an allelic genetic model . No significant increased CAD risk was detected in subgroup 1 under an allelic genetic model . Studies with a total size of >200 were included in subgroup 1, and the residual studies with a total size of <200 were included in subgroup 2 .The publication bias of the studies was assessed using the funnel plot and Egger’s test. Publication bias was not seen in the funnel plot . No statcontrols. Significant association was observed between PAI-1 4G/5G gene polymorphism and CAD under an allelic genetic model in the Chinese Han population (OR = 1.70). This finding suggested that the carriers with the 4G allele of the PAI-1 4G/5G gene in the Chinese Han population might be predisposed to CAD.The current meta-analysis included 879 CAD patients and 628PAI-1 4G/5G gene polymorphism with CAD was evidently weakened in subgroup 1 and not yet significant (OR = 1.56). In contrast, the association of PAI-1 4G/5G gene polymorphism with CAD was evidently strengthened in subgroup 2 (OR = 2.03). Hence, further studies that can provide a match between the sample sizes of cases and controls are warranted.In the subsequent meta-regression, the confounding factors  could explain the heterogeneity. This result suggested that the non-uniformity in the abovementioned factors contributed to the heterogeneity among the individual studies. In a stratified analysis by the total size, significantly increased risk was only detected in subgroup 2 under an allelic genetic model. After adjusting for the total sample size, unexplained heterogeneity still existed. The association of PAI-1 is a glycoprotein that belongs to the serine protease inhibitor superfamily. It is equimolecularly combined with the tissue plasminogen activator (tPA) single chain, double chains, and double chain urokinase plasminogen activator (uPA). Consequently, tPA and uPA activities are rapidly inhibited by PAI-1. PAI-1 is also an essential regulatory substance for in vivo plasminogen activation. PAI-1, which is synthesized by endothelial cells and the liver, is regulated by hormones and neurotransmitters. A dose-response relationship has been found between PAI-1 and such metabolism syndrome-related factors as obesity, hypertriglyceridemia, low high-density lipoprotein hyperlipidemia, hyperglycemia, hyperinsulinemia, and physical labor deficiency PAI-1 4G/5G gene polymorphism is one of the DNA sequence variations that plays a key role in regulating PAI-1 gene expression. Studies have shown that the PAI-1 activity of the 4G allele promoter is higher than that of 5G in a cytokine-stimulated state. PAI-1 4G/5G gene polymorphism also influences PAI-1 gene transcription similarly in non-stimulated cells G or 5G allele could be combined with protein A, which is a transcription activator. The 5G allele could be incorporated with protein B, which hinders the combination of the transcription activator. The action locus is certified near the alleles. The 4G homozygote lacks the combination site with B factor, thus the 4G homozygote could not be combined with protein B, which contributes to elevated plasma PAI-1 activity PAI-1 4G/5G gene polymorphism and CAD risk is subject to much controversy worldwide. In 2003, Böttiger C et al. reported that 4G/5G polymorphism of the PAI-1 gene is not associated with an increased risk of thrombotic and restenotic events after coronary artery stenting in Germany PAI-1 gene polymorphisms in young adults in Brazil 4G/5G polymorphism is observed in MI risk in France PAI-1 4G/4G genotype is an independent predictor for the development of MI in a Turkish population The relationship between PAI-1 4G/5G gene polymorphism and CAD are still inadequate. PAI-1 is influenced not only by PAI-1 4G/5G gene polymorphism, but also by environmental factors, such as the concentration of blood sugar, insulin, triglycerides, and so on The present research has some limitations. Large-scale studies on the relationship between G allele of PAI-1 4G/5G gene polymorphism might increase the CAD risk in the Chinese Han population. Therefore, significant potential guidance for formulating CAD individual therapies is provided. Taking into account the aforementioned limitations, further studies are highly needed in the future.The current meta-analysis indicated that the 4File S1PRISMA 2009 Checklist.(DOC)Click here for additional data file.File S2PRISMA 2009 Flow Diagram.(DOC)Click here for additional data file.


CT + CC [p 0.0081 OR 3.4 95%CI (1.27-11.9)] and T vs. C allele [p 0.02 OR 1.4 95%CI (1.03-1.9)]. Our data thus confirm the association between the rs7903146 T allele and T2D in an Iranian population, contrary to previous reports in Arab populations. This can possibly be attributed to differences in ethnic background or the effects of environmental factors.The transcription factor 7-like 2 gene (TCF7L2) rs7903146 T allele is constantly associated with Type 2 diabetes in various populations and ethnic groups. Nevertheless, this has not been observed in two studies involving Arab populations. The aim of the present study was to investigate the association between TCF7L2 rs7903146 in an Iranian population. Type 2 diabetes patients (N = 258) and normal healthy control subjects (N = 168) from the same area, were examined. The ARMS- PCR (Amplification Refractory Mutation System) technique, subsequently validated by direct sequencing, was used for genotyping. Allele and genotype frequencies were significantly different between patients and controls TT  Type 2 diabetes (T2D) is a common complex disorder resulting from the interplay of genetic and environmental factors . FollowiOther genetic loci, including PPARG and KCNJ11, have also been reproducibly associated with type 2 diabetes. More recently, 16 novel T2D susceptible loci have been identified  was employed for genotyping. The following primers were used:Common forward primer: 5' - ggagc cgtca gatgg taatg -3'TCF4-C: 5' - ggtg cctca tacgg caatt aaatt atatag -3'TCF4-T: 5' - ggtg cctca tacgg caatt aaatt atataa -3'β-actin- F: 5' - ctt cct tcc tgg gca tgg ag -3'β-actin- R: 5' - tgg agg ggc cgg act cgt ca -3'The PCR cycles were as follows: 5 min at 95 °C, followed by 35 cycles of 30 s at 95°, 30 s at 66 °C and 60 s at 72 °C. Final extension was 7 min at 72 °C. The presence of PCR product (188 bp for TCF7L2 and 315 bp for β-actin) was verified on 2% agarose gel stained with ethidium bromide. Genotyping results were validated by direct sequencing.Odd ratios (OR) and 95% confidence intervals (CI) were used for estimating the strength of association between different groups and alleles or genotypes of TCF7L2 gene polymorphism. Significance levels were defined through either Chi-quare or Fisher exact analyses with contingency tables. All analyses were carried out using STATA (v8) software.Genotype frequencies did not differ from the expected Hardy-Weinberg ratios. The Male/Female ratio was 68/170 in diabetic patients and 58/72 in controls. The mean age was 53 ± 10 in diabetic patients and 52 ± 10 in normal controls. The mean BMI was 28 ± 4 in patients with diabetes and 26 ± 3 in normal controls.vs. CT +CC [p 0.0081 OR 3.4 95%CI(1.27-11.9)] and T vs. C allele [p 0.02 OR 1.4 95%CI (1.03-1.9)] Allele and genotype frequencies for TCF7L2 rs7903146 variation was significantly different in cases compared to the controls TT in vivo and in vitro  box-containing transcription factor which is part of the Wnt signaling pathway (In most recently published studies, it has been suggested that TCF7L2 is associated with impaired insulin secretion, but not with increased insulin resistance (Our data confirm the association of this widely replicated variant with T2D risk in Iranians, in contrast with previous reports in Arab populations. Differences in ethnic background or the effect of environmental factors, such as life-style, may explain these discrepancies.


SOD2 gene Val16Ala polymorphism and albuminuria, but this association has not been explored in the Mexican population.Several studies in type 2 diabetes patients have shown significant associations between the SOD2 gene Val16Ala polymorphism (rs4880) and macroalbuminuria in a sample of 994 unrelated Mexican type 2 diabetes patients. The study included 119 subjects with urinary albumin >300 mg/dL and 875 subjects with urinary albumin ≤ 30 mg/dL. Genotyping of the SOD2 gene Val16Ala SNP was carried out with Real-Time Polymerase Chain Reaction (RT-PCR).We evaluated the association between the The frequency of the TT genotype was 6.7% higher in participants with macroalbuminuria than in the normoalbuminuria group (16.8% vs. 10.1%). Using a logistic regression analysis, we observed that individuals with the CC genotype had significantly lower risks of macroalbuminuria than those with the TT genotype . We carried out a meta-analysis combining our data with data from four previous studies and estimated an odds ratio (95% CI) for the C allele  of 0.65 .SOD2 Val16Ala polymorphism and macroalbuminuria in a sample of Mexican type 2 diabetes patients.A significant association was found between the  Diabetic nephropathy (DN) is the leading cause of chronic kidney disease . HoweverSOD2 gene has been predicted to cause a conformational change in the target sequence, from a partial alpha-helix in the Ala-variant  to a beta-sheet in the Val-variant , which induces a 30-40% decrease in SOD2 activity due less efficient transport of the protein into the mitochondrial matrix [Superoxide dismutase 2 (SOD2), also known as manganese superoxide dismutase (MnSOD), is one of the major antioxidant defense systems against mitochondrial superoxide radicals . The Vall matrix ,9.Population differences in the incidence and progression of renal diseases have been described previously ,11. NatiSOD2 gene Val16Ala polymorphism and albuminuria, but this association has not been explored in the Mexican population. The aim of the study was to assess the association of the SOD2 gene Val16Ala polymorphism with macroalbuminuria in a sample of T2D patients from Mexico City.Several studies in type 2 diabetes (T2D) patients of Japanese, Korean and Chinese origin -18, as wThe project was approved by the 3609 Local Ethics Committee of the National Institute of Social Security (IMSS) according to the declaration of Helsinki. Written informed consent was obtained from each participant.The study included 994 unrelated Mexican T2D patients recruited from primary care clinics of the IMSS, in Mexico City, between 2008 and 2010. Diabetes diagnosis was done according to the American Diabetes Association criteria, i.e. fasting plasma glucose ≥7.0 mmol/l or OGTT ≥11.1 mmol/l for the 2 h sample . None ofDNA was extracted from peripheral blood using the QIAamp  kit following the manufacturer’s recommendations. DNA concentration was measured by optical density , and integrity by electrophoresis in 0.8% agarose gels stained with ethidium bromide .SOD2 gene Val16Ala polymorphism was genotyped with RT-PCR, using the TaqMan® SNP Genotyping Assay .The Individual admixture proportions were available for 386 out of the 875 participants with normoalbuminuria, and for 53 of 119 subjects in the macroalbuminuria group. For these individuals, Native American, European and West African contributions were estimated with the program ADMIXTURE , using ahttp://ihg.gsf.de/cgi-bin/hw/hwa1.pl).Differences between groups were assessed using t-Student and chi-square tests for numerical and categorical variables, respectively. Deviations from the Hardy-Weinberg proportions were evaluated using an exact test  or T2D duration. Finally, an inverse variance meta-analysis including this study as well as previously published studies [http://www.sergas.es/MostrarContidos_N3_T01.aspx?IdPaxina=62715).In order to test if the  studies -18 was cA total of 119 participants with macroalbuminuria and 875 with normoalbuminuria were included in the study. Table SOD2 gene Val16Ala polymorphism in the comparison groups. There were no significant deviations from Hardy-Weinberg proportions in the normoalbuminuria or macroalbuminuria groups. The frequency of the T allele was higher in the macroalbuminuria group than in the normoalbuminuria group (41.6% vs. 32.9%). With respect to the genotypes, the frequency of the TT genotype was 6.7% higher in the participants with macroalbuminuria than in the normoalbuminuric subjects (16.8% vs. 10.1%). The effect of the SOD2 gene Val16Ala polymorphism on macroalbuminuria was evaluated using a logistic regression model including sex, duration of T2D, body mass index, systolic blood pressure, diastolic blood pressure, current or previous smoking, HbA1c, total cholesterol, HDL-cholesterol, LDL-cholesterol, previous diagnosis of hypertension and hypertension treatment as covariates . Table Table We carried out an inverse variance meta-analysis including our study and four additional studies for which genotype data are available. We report the results using an allele model (C vs. T), but other models (e.g. codominant or dominant TT vs. CC/CT) give consistent results. Table SOD2 gene, on DN risk. A number of studies have focused on the SOD2 gene Val16Ala polymorphism, which is a non-synonymous polymorphism that has been associated with DN in several populations. However, although polymorphisms in the ACE [SOD2 gene Val16Ala polymorphism and macroalbuminuria has not been yet studied in Mexico.Diabetic nephropathy is an important public health problem in Mexico. DN is the leading cause of end stage renal disease in adults and is responsible for approximately 75% of the total costs associated with the treatment of diabetic complications . Previou the ACE  and TGF- the ACE  genes haSOD2 gene Val16Ala polymorphism in 994 T2D patients: 119 individuals with macroalbuminuria and 875 normoalbuminuric subjects. In a logistic regression model incorporating other relevant covariates, the CT and CC genotypes had lower odds ratios than the reference TT genotype . Given the different odd ratios observed for the CT and CC genotypes, a codominant model (CC vs. CT vs. TT) seems to provide the best fit to the data. Using a dominant model, instead of an unconstrained model, the CC+CT genotypes had an OR=0.55 (p=0.026), with respect to the TT genotype.We genotyped the SOD2 gene Val16Ala polymorphism with macroalbuminuria. Our results are consistent with a growing number of studies in different population groups [SOD2 Val16Ala C allele, which codes for the amino acid alanine, confers a protective effect against macroalbuminuria. This protective effect has been explained as a result of the conformational change caused by alanine, which increases the efficiency of protein targeting to the mitochondria, thereby leading to a rise of reactive oxygen species scavenging in the mitochondria, and a reduction in glomerular membrane damage due to oxidative stress [To our knowledge, ours is the largest study to date evaluating the association of the n groups -19, and e stress ,9.The contemporary Mexican population is the result of an admixture process that involved Native American, European and African populations. It is well known that the presence of population stratification can inflate the rate of false positives in association studies in admixed populations . For exaWhen we restricted our analysis to the participants for which admixture information was available, we observed that macroalbuminuria subjects had a lower Native American ancestry than normoalbuminuria group . The odds ratios of the logistic regression analysis incorporating ancestry in the model are similar to those observed in the full sample, again indicating that the CC and CT genotypes have a protective effect against macroalbuminuria . The p-values obtained in this analysis are slightly weaker than in the full sample, but this is not surprising considering that the sample size was reduced in half, with a subsequent reduction in power to identify significant effects. Overall, our data suggests that the significant results observed in the analysis of the full dataset are not due to the presence of population stratification. If stratification was responsible for the results observed in the full sample, we would expect that an analysis of the subsample incorporating ancestry in the statistical models would lead to differences in the ORs observed in both samples. Instead, the ORs are similar in both analyses . We also estimated the odds ratios restricting the normoalbuminuric sample to subjects with more than 10 years of duration of T2D and the results are very similar to those observed in the full sample.SOD2 Val16Ala polymorphism risk allele T, which codes for the amino acid valine, in the normoalbuminuric group from Mexico City is quite similar to the frequencies observed in the HapMap Mexican American sample from Los Angeles, California (32.9% vs. 36.0%) (http://hapmap.ncbi.nlm.nih.gov/cgi-perl/snp_details_phase3?name=rs4880&source=hapmap28_B36&tmpl=snp_details_phase3). However, the data available for other population groups indicate that there is substantial dispersion of allele frequencies for this polymorphism. For example, in populations from East Asia the frequencies of the T allele are higher than 85%, and in populations of African and European ancestry, the T allele has intermediate frequencies, between 50% and 65%.The allelic frequency of the SOD2 Val16Ala polymorphism to a model including the aforementioned factors increased only slightly the goodness of fit of the model . Some of the factors identified in our study  have also been highlighted in previous studies [In our logistic regression analyses, we included a number of covariates that were significantly associated with macroalbuminuria in our sample . We observed that adding the  studies ,30. Howe studies -32, alth studies ,34. Unfo studies , and in  studies .SOD2 gene Val16Ala polymorphism is associated with macroalbuminuria. Our results highlight the importance of exploring the effects of polymorphisms located within genes relevant to oxidative stress in order to understand the genetic basis of diabetes complications.In summary, we show that the ACE: Angiotensin converting enzyme; AFR: African; AIMs: Ancestry Informative Markers; ARB: Angiotensin receptor blockers; CI: Confidence intervals; DN: Diabetic nephropathy; EUR: European; Hb1Ac: Glycated hemoglobin; IMSS: National Institute of Social Security; MnSOD: Manganese superoxide dismutase; NAM: Native American; RT-PCR: Real-time polymerase chain reaction; SOD2: Superoxide dismutase 2; T2D: Type 2 diabetes.The authors declare that they have no competing interests.IJAM, EJP, AVS, JKR and MC conceived and designed the study. IJAM, AVS, JEP and JHGZ acquired the data. IJAM, EJP, AVS, MC interpreted the data. All authors revised the manuscript for important intellectual content, read, and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/14/110/prepub


NFκB1 (-94del/insATTG) and NFκBIA (2758 A>G) polymorphisms were associated with colorectal cancer (CRC) susceptibility.Nuclear factor κB (NFκB) plays a key role in the regulation of apoptosis. The function of NFκB is inhibited by binding to NFκB inhibitor (IκB), and disruption of the balance of NFκB and IκB is related to the development of many diseases, including tumors. Therefore, we hypothesized that the NFκBIA alter its gene expressions and functions and thus cancer risk.In a hospital-based case–control study of 1001 CRC patients and 1005 cancer-free controls frequency matched by age and sex, we genotyped polymorphisms using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and performed luciferase assays and Western blotting analysis to identify whether genetic variants in NFκB1-94 ins/delATTG and NFκBIA 2758 A>G polymorphisms were correlated with CRC risk . Furthermore, when evaluated these two polymorphisms together, the combined genotypes with 2 variant (risk) alleles (2758GG and -94ins/ins+del/ins) were associated with an increased risk of CRC  compared to 0 variant, and the significant trend for 2 variant (risk) alleles were more pronounced among subgroups of aged <60 years, women, never drinkers, never smokers, persons with a normal BMI and those with a family history of cancer(trendP<0.01). Moreover, luciferase assays showed that the G allele in the 3′UTR significantly decreased NFκBIA mRNA stability and the A allele regulation by miRNA449a in vitro and that the NFκBIA protein expression levels of the AA+AG variant carriers were significantly higher in peritumoral tissues than those of the 2758GG genotype.We found that both NFκB1 and NFκBIA polymorphisms appear to jointly contribute to risk of CRC. These two variants may be a genetic modifier for CRC susceptibility in this southern Chinese population. As the NFκB/IκB system plays an important regulatory role in the apoptotic pathway and dysregulated expression of the NFκB1 and NFκBIA has been observed in CRC, we hypothesized that combined NFκB1 and NFκBIA polymorphisms may be associated with increased risk of CRC. To test this hypothesis, we genotyped the NFκB1-94 insertion/deletionATTG and NFκBIA 2758A>G polymorphisms in our ongoing hospital-based case–control study of CRC in a southern Chinese population, and further performed luciferase assays and Western blotting analysis to identify whether genetic variants in NFκBIA alter its gene expressions and functions and thus cancer risk.There has been no previous report on the association between The study protocol was approved by the institutional review boards of Sun Yat-Sen University. Written informed consent was obtained from each participant after a full explanation of the study.From July 2002 to April 2010, a total of 1001 patients with histopathologically-confirmed and untreated sporadic CRC were prospectively recruited from the First and Sixth Affiliated Hospital  and Cancer Center of Sun Yat-Sen University, Guangzhou, China, the Affiliated Tumor Hospital of Guangzhou Medical College, Guangzhou, China, Guangdong Provincial People's Hospital, Guangzhou, China, and Panyu People's Hospital, Guangzhou, China. All these subjects were genetically unrelated ethnic Han Chinese and were from the city of Guangzhou and surrounding regions in southern China. Of the 1001 cases included in this study, there were 169 (16.9%) cases of right colon cancer, 309(30.9%)cases of left colon cancer, and 523 (52.2%) rectal cancer. According to American Joint Committee on Cancer staging Manual, there were 171 (17.1%)cases of stage I, 320 (31.9%)stage II, 345 (34.5%)stage III, and 165 (16.5%)stage IV. In the interim, a total of 1005 cancer-free controls were randomly selected from a subject pool of more than 10,000 individuals who participated in health check-up programs at the community health stations in Guangzhou, China.2 were categorized as being underweight, subjects whose BMI was from 18.5 to 24.0 kg/m2 were normal body weight, those who have a BMI >24.0 kg/m2 were overweight The study participants were interviewed and data on smoking status, alcohol use and other factors including family history of cancer were obtained using a structured questionnaire. Smoking status, alcohol use and family history of cancer were defined as described previously NFκB1 promoter (rs28362491) polymorphism, the SNP-containing fragment was amplified using the following primers: 5′-TGGGCACAAGTCGTTTATGA-3′ (forward) and 5′-CTGGAGCCGGTAGGGAAG-3′ (reverse). PCR was run at 94°C for 3 min followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s with a final extension at 72°C for 10 min. The PCR products (281/285 bp in size) were digested with PfIMI  at 37°C overnight followed by 2% agarose gel electrophoresis. For determination of the NFκBIA (rs696) polymorphism, the SNP-containing fragment was amplified using the following primers: 5′-GGCTGAAAGAACATGGACTTG-3′ (forward) and 5′-GTACACCATTTACAGGAGGG -3′ (reverse). The PCR was run at 94°C for 5 min followed by 32 cycles of 94°C for 30 s, 54.3°C for 45 s and 72°C for 60 s with a final extension at 72°C for 10 min. The amplified fragments were digested with HaeIII  overnight at 37°C followed by 2% agarose gel electrophoresis.Five mL blood was collected from each participant and genomic DNA was extracted using the DNA Blood Mini Kit  according to the manufacturer's instructions. Genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. For determination of the Genotype analysis was done by two experimenters independently who were blinded to the status of the subjects as patient or control. To further validate the genotyping results, we randomly selected 10% samples for each of the 2 SNPs to perform repeat assays, and the results were 100% concordant. Additionally, 5% of the PCR products for each target genotype were purified and confirmed by direct sequencing .NFκBIA could bind to microRNAs (miRNA), we searched for target microRNAs of genetic variants of NFκBIA using the algorithm programs (http://www.targetscan.org and http://microrna.sanger.ac.uk/cgi-bin/targets/v5/search.pl).To investigate whether genetic variants of Three human colorectal adenocarcinoma cell lines, HCT116, HT29 and SW480 were purchased from Culture Collection of Chinese Academy of Science  and routinely cultured in Dulbecco's Modified Eagle's Medium or RPMI 1640 medium supplemented with 100 units/ml of penicillin, 100 µg/ml of streptomycin, and 10% fetal bovine serum (FBS). The cells were grown at 37°C with 5% CO2 in a humidified incubator.MiR-449a mimics targeting NFκBIA, 5′-UGGCAGUGUAUUGUUAGCUGGU-3′ (sense) and 5′-CAGCUAACAAUACACUGCCAUU-3′ (anti-sense), miR-449a inhibitor, 5′-ACCAGCUAACAAUACACUGCCA-3′, miR-34b mimics targeting NFκBIA, 5′-CAAUCACUAACUCCACUGCCAU-3′ (sense) and 5′-GGCAGUGGAGUUAGUGAUUGUU-3′ (anti-sense), and miR-34b inhibitor, 5′-AUGGCAGUGGAGUUAGUGAUUG-3′ were synthesized by GenePharma Co. . In addition, the 3′UTR of the NFκBIA 2758A allele (2575 to 2955 bp relative to the translation start site) containing the miR-449a and miR-34b binding site was amplified using the following two primers, 5′-CCGCTCGAGCAAAGGGGCTGAAAGAA-3′ (sense) and 5′-AAGGAAAAAAGCGGCCGCAAAATGTGGTCCTTCCATGA-3′ (anti-sense). The amplified fragments were restricted with XhoI and NotI  and then ligated into the XhoI and NotI restriction site of the dual-luciferase plasmid, psiCHECK™-2 . Additionally, the 3′-UTR of the NFκBIA 2758G allele, which contained a mutated mRNA binding site, was amplified using PCR site-directed mutagenesis and cloned into psiCHECK™-2. For transfection, cells were seeded onto 24-well plates at 1×105 cells/well and, after an overnight incubation, the cells were co-transfected with the reporter plasmid  and miR mimics or inhibitors using Lipofectamine 2000  following the manufacturer's protocol. Cells were lysed 48 h after transfection, and luciferase activity was measured using a Dual-Luciferase Reporter Assay System . Each experiment was done in triplicate and at least three times independently.NFκBIA polymorphisms in its 3′UTR (2758A>G) and NFκBIA protein expression levels in tissues, 32 paired tumor and peritumoral tissues were obtained from sporadic CRC patients resected at the Sixth Affiliated Hospital of Sun Yat-Sen University and archived in the tumor bank. All tissues samples were histologically confirmed. Immunoblotting assays were performed as previously described To analyze the correlation between P<0.05 was considered statistically significant.Two-sided chi-square tests were used to assess differences in the distributions of age, sex, smoking status, alcohol use, BMI, menstruation history, and family history of cancer between cases and controls. The Hardy-Weinberg equilibrium (HWE) was tested by a goodness-of-fit chi-square test to compare the expected genotype frequencies with observed genotype frequencies (p2+2pq+q2 = 1) in cancer-free controls. The association between case-control status and each SNP, measured by the odds ratio (OR) and its corresponding 95% confidence interval (CI), was estimated using an unconditional logistic regression model, with and without adjustment for age, sex, smoking status, alcohol drinking status, BMI, tumor site, and family history of cancer. Recent studies indicate that an analysis of the combined genotypes might be more scientifically significant than an analysis of a single polymorphism in predicting the disease associations. Therefore the combined genotype data were further stratified by age, sex, and smoking status, alcohol drinking status, BMI, tumor site, and family history of cancer. Logistic regression modeling was also used for the trend test. Student's t test was done to examine the difference in levels of luciferase reporter gene expression between different constructs. Kruskal-Wallis one-way ANOVA tests were used for analyzing NFκBIA protein expression in peritumoral tissues of different genotypes. All tests were two-sided by using the SAS software . P>0.05 in all). Compared with controls, there were more ever drinkers  (P<0.01) in the cohort of CRC cases. Moreover, compared with controls, CRC cases were more likely to have a family history of cancer or a higher BMI (P<0.05 in both). Consequently, these variables were further adjusted for in the multivariate logistic regression model to avoid possible confounding on the main effects of the SNPs under study. They were also used in the subsequent stratification and gene-environment interaction analysis.The distribution of demographic characteristics of the 1001 sporadic CRC cases and 1005 cancer-free controls are shown in NFκB1 promoter region by the PCR-RFLP method. Two ATTG repeats are present at the NFκB1 promoter region, and one allele that has an ATTG insertion (ins) was cleaved into a 45-bp and a 240-bp fragment after digestion with PfIMI while the other deletion allele (del) that has only one ATTG at its promoter was not cleaved  and NFκBIA 2758A>G (rs696) polymorphisms among the cases and controls are summarized in P>0.05). As shown in P<0.01; del/ins: OR = 1.33; 95% CI = 1.03–1.74; P<0.01; ins/ins+del/ins: OR = 1.47; 95% CI = 1.14–1.86; P<0.01, respectively). For rs696, there was a significant difference in the distribution of the rs696 genotypes between CRC cases and controls (P<0.01). Significant differences were also noted in the distribution of the recessive model of rs696 (GG versus AA+AG) between CRC cases and controls . Consistently, significant association was found between the two SNPs and the risk of sporadic CRC.The genotype and allele distributions of the NFκB1 and NFκBIA polymorphisms by examining age, sex, smoking, drinking, BMI, tumor site, and family history of cancer by logistic regression. We combined the NFκB1 and NFκBIA polymorphisms based on the number of variant (risk) alleles . As shown in NFκB1-94 del/del and NFκB1IA 2758AA+AG genotype, one variant combined genotype carriers who were <60 years of age had a higher risk of CRC  (P<0.05). Further stratification analysis revealed that individuals with two variants had a significantly higher risk of CRC among subgroups of patients aged <60 years  (P<0.01), women  (P<0.01), never smokers  (P<0.01), never drinkers  (P<0.01), persons with a normal BMI  (P<0.01) and those with a family history of cancer .We further analyzed the combined genotypes of the NFκBIA 2758 A>G polymorphism using a computer algorithm and found that the polymorphism could affect miRNA binding. MiR-449a and miR-34b, which are involved in a wide variety of biological functions, were found to have a binding site within the 3′-UTR of the NFκBIA 2758 A>G polymorphism  . Taken together, these results demonstrated that the 2758G allele in the 3′UTR of the NFκBIA reduced normalized luciferase activity compared to the 2758A allele, likely corresponding to reduced mRNA stability or translational efficacy and that miR-449a have the ability to bind and partially repress luciferase expression via the NFκBIA 3′UTR segment when carrying the 2758A allele of NFκBIA 2758 A>G polymorphism.We further analyzed the 3′UTR of the morphism . To detehibitors . We foun( P<0.05). MiR-449 HCT116) . On the NFκBIA3′UTR polymorphisms (2758A>G) were associated with increased or reduced expression of NFκBIA protein. We collected 32 paired tumor and peritumoral tissues from the untreated CRC patients of different genotypes. Immunoblotting analysis revealed that the levels of NFκBIA (NFκBIA/β-actin protein ratio) in the peritumoral tissues of 12 cases of the 2758AA genotype was 0.80±0.09 and those of 8 cases of the 2758AG genotype was 0.63±0.11, both of which were significantly higher than those of the other 12 cases of the 2758GG genotype (0.47±0.06)   . HoweverNFκB1-94del/insATTG and NFκB1IA 2758A>G polymorphisms with risk of CRC in a southern Han Chinese population. We found that both the NFkB1-94del/ins ATTG and NFkBIA 2758A>G polymorphisms were associated with increased risk of CRC. For the NFkB1-94del/insATTG polymorphism, with the -94del/del as the reference, we found that the -94(ins/ins+del/ins) genotype was associated with a statistically significantly increased risk of CRC. For the NFKBIA 2758A>G polymorphism, with the 2758(AA+GA) as the reference, we also found that the 2758GG genotype was associated with a statistically significantly increased risk of CRC. Furthermore, when we evaluated NFkB1 and NFKBIA polymorphisms in combination, we found that the combined 2758GG and -94ins/ins+del/ins genotype was associated with a significantly increased risk of CRC compared with those without the 2758GG and -94ins/ins+del/ins genotype, and this increased risk was more pronounced among younger than 60 years, women, never drinkers, never smokers, persons with a normal BMI and those with family history of cancer. In the in vitro assays, we also found that, compared with the 2758A allele, the 2758G variant allele showed significantly decreased mRNA stability and/or translational efficacy. Furthermore, we found that NFkBIA levels significantly higher in peritumoral tissues from patients of the 2758AA+AG genotypes than those of the 2758GG genotypes, suggesting that the 2758A>G polymorphism is potentially functional and the polymorphism 2758A>G at 3′UTR of NFkBIA could affect gene expression. To the best of our knowledge, this is the first study to investigate whether NFkB1 and NFkBIA polymorphism and their combined polymorphism were associated with risk of CRC.In the present study, we investigated the associations of the NFκB1 pathways seem to play a critical role in multiple human pathologies by regulating the transcription of genes involved in the immune response, cell proliferation, and apoptosis NFκB1 expression plays an important role in the protection of cells from apoptosis NFκB1 activity has been observed in various types of cancer NFκB1 and NFκBIA genes, if functional, could be expected to have an effect on cell death, and thus, carcinogenesis.There are several lines of evidence supporting our findings. The NFκB1 and NFκBIA polymorphisms is related to the development of inflammatory and other diseases including ulcerative colitis, Graves' disease, and diabetes mellitus, and susceptibility to tumors including melanoma, bladder cancer and CRC in different ethnic groups et al.'s study reported a lack of an association between the NFκBIA2758 A>G polymorphism in northern Chinese population, but showed an association with the Swedish population and CRC risk del allele may result in relatively decreased NFκB1 transcript levels and hence decreased p50/p105 NFκB1 protein production 2758 A to G allele in the 3′UTR of NFκBIA decreased luciferase activities as assessed by luciferase assays. Our functional in vitro experiments suggested that NFκBIA 2758 A>G variants may affect mRNA stability. However, that the NFκBIA 2758 A>G variants affects translational efficacy or conduces to differential nuclear RNA processing or export also cannot be completely excluded NFκBIA generates a potential seed site for miR-449a and our ex vivo luciferase data indicated that miR-449a reduced the relative luciferase activities via the NFκBIA 3′UTR target site created by the 2758A allele. The results indicated that the 2758A allele strengthens the binding of miR-449a with 3′UTR of NFκBIA, which in turn inhibits the expression of NFκBIA. Recent evidence indicate that miRNAs can bind to the 3′ UTRs of mRNAs and affect their translation, thus regulating cell proliferation, apoptosis and tumorigenesis NFκBIA 2758A-allele may enhance binding of miR-449a and affect NFκBIA gene expression and change of the 2758 A to G allele in the 3′UTR of NFκBIA may affect mRNA stability or translational efficacy subsequent diminished protein levels. Consistently, we observed that NFκBIA protein expression was higher in the peritumoral tissues but not in tumor tissues from patients of the 2758AA+GG genotype than those of the 2758GG genotype. These data suggest that wildtype A allele may result in overexpression of NFκBIA in peritumoral normal tissues and hence increased NFκBIA protein production, resulting in inhibition of NFκB activity. However, carrying a mutant 2758G allele result in relatively decreased NFκBIA mRNA stability and hence diminished NFκBIA protein production. As a major inhibitor of NFκB, decreased expression and dysfunction of NFκBIA may be a direct result of the activation of NFκB and thus cancer NFκBIA G allele. These findings indicate that the NFκB1 -94del/ins ATTG and NFκBIA 2758 A>G polymorphisms are functional. Our data from this relatively large sample size study further support the notion that the NFκB1 and NFκBIA polymorphisms are potentially implicated in cancer risk.Several association studies have reported that the NFκB1 and NFκBIA polymorphisms on risk of CRC was more pronounced among younger than 60 years, women, never drinkers, never smokers, persons with a normal BMI and those with a family history of cancer, suggesting that, in these subpopulations, gene-environment interaction may be very weak and the combined effect was an independent risk factor for these subpopulations. Compared with the published data, our results indicate that the genotype distributions of the NFκB1 and NFκBIA polymorphisms vary with ethnicity. For example, the frequencies of the del/del, del/ins, and ins/ins genotypes of the NFκB1-94del/insATTG among our 1005 southern Han Chinese control subjects were 18.5%, 51.9%, and 29.6%, respectively, compared with 15.6%, 45.9%, and 38.4%, respectively, of 307 Germans in the study by Kathrin et al.AA, AG and GG genotypes of the NFκBIA 2758A>G in our controls were 21.2%, 52.8%, and 26.1%, respectively, compared with 6%, 45%, and 49%, respectively, of 109 cancer-free Australian controls in the study by Curran et al.del/del, del/ins, and ins/ins genotypes of the NFκB1-94del/insATTG  were very similar to the published data by Sun et al.  NFκBIA genotype distributions is also similar to that reported for that of the northern Han Chinese insertion of NFκB1-94del/insATTG polymorphism increased CRC risk, which was contrary to the results reported by Andersen et alIn the present study, we also observed that the combined effect of the NFκB1 and NFκBIA polymorphisms have effect on risk of CRC. These findings suggest that the NFκB1 and NFκBIA polymorphisms may jointly contribute to the risk of CRC in a southern Chinese population, which were consistent with the functional assays we performed. Our study indicated that the NFκB1-94(ins/ins+del/ins) and NFκBIA GG polymorphism may be a genetic marker for susceptibility to CRC in Chinese populations. However, additional studies with more detailed data on environmental exposure and survival data are required to verify these findings. Therefore, future population-based studies are needed to verify the findings.In conclusion, our results suggested that both Figure S1NFκB1-94ins/delATTG genotyping by direct sequencing: (a) del/del genotype; (b) del/ins genotype; (c) ins/ins genotype.(PPT)Click here for additional data file.Figure S2NFκBIA 2758A>G genotyping by direct sequencing: (a) GG genotype; (b) AA genotype; (c) AG genotype.(PPT)Click here for additional data file.