Epidemics of HFMD are elevated every year globally, especially in mainland China. The disease now presents as an increasing threat to public health worldwide.Five hundred and seventy-one EV71-infected HFMD patients in Beijing You'an Hospital were grouped by disease severity: Mild (no severe complication) (n = 221), and Severe group , and/or pulmonary edema (PE) (n = 350)). Clinical and laboratory findings and levels of 7 serum cytokines were analyzed.9/L (p = 0.040) were associated with severe cases. Results of multivariate analysis indicated five independent risk factors (RR>26/min (p<0.001), Age<4 yo (p<0.001), GLU>8.3 mmol/L (p = 0.011), LYM>40% (p = 0.010), and ALT>40 U/L (p = 0.045)). In addition to single-factor analysis, we further analyzed the use of different combinations of risk factors. “GLU>8.3 and CL<98 and RR>26” (confidence ration (CR) = 100%) is the top indicator, followed by “ALT>40 and LYM>40% and RR>26 and Age<4 yo” (CR = 92.9%).Univariate analysis showed that (RR)>26/min (p<0.001), age<4 yo (p = 0.001), GLU>8.3 mmol/L (p = 0.008), CL<98 mmol/L (p = 0.026), and WBC>1.2×10Serum levels of IL-2, IL-4, IL-10, IFNγ, GM-CSF, and TNFα were higher in severe cases than in mild cases. A new evaluation scoring system by scoring each risk factor 1 and independent risk factor 2 was developed for early identification of severe HFMD cases.Five independent risk factors, along with indicative combinations of risk factors, for severe cases were identified, and a scoring system was created to facilitate the use of indicators for early medical intervention. Enterovirus genus in the Picornavirus family. Although HFMD could be seen globally, Mainland China is one of the main regions where EV71 outbreaks could be seen, in almost every year recentlyHand, foot and mouth disease (HFMD) is a common disorder in young children. It is considered as a potentially life-threatening diseaseAccumulating evidence from global reports on HFMD epidemics supports the fact that the ratio of severe cases is elevating gradually, along with mortality rateThe objective of this study was to analyze the clinical and laboratory data of a group of pediatric HFMD patients admitted to Beijing You'an Hospital, Capital Medical University, and evaluate the correlations between early clinical-laboratory findings and disease severity. This study aims to identify early indicators of disease severity so that prophylactic measures can be taken to reduce mortality. In addition to evaluate individual markers, we tried to use different combinations of markers as indicators of severity.The case definition is described elsewhereInformation of the study population consisted of 571 children who met the case definition described above was collected retrospectively. The patients were consecutively admitted to Beijing You'an Hospital, Capital Medical University  between Mar and Oct, 2012. All these cases were tested for serum cytokine levels.Patients were divided into two categories based on severity: (1) Mild: patients with HFMD without BE or PE; (2) Severe: HFMD patients complicated by BE or/and PE.All of the parameters included in the investigation were collected by reviewing the patient medical records that were preserved in the medical record library and the medical computerized database at Beijing You'an Hospital, Capital Medical University. The patient records were retrospectively examined for primary set of data, which included demographic characteristics (age and sex), clinical parameters (signs and symptoms), laboratory values , radiologic data, outcome at discharge (recovered or died), admission and discharge dates, and length of hospital stay. Clinical findings and laboratory results analyzed in this study were from the examination done right upon admission of all involved patients.You'an Hospital Ethics Committee has approved this study. It covered the retrospective analysis of the 571 records. Parents or caretakers of all 571 participants have given a written informed consent on behalf of the children participants for their information to be stored and used for research. Human experimentation guidelines of PR.China were followed in the conduct of this clinical research.The blood samples for cytokine determination were collected immediately upon patient admission. The plasma was harvested within 30 min at 37°C of venipuncture from EDTA-anticoagulated blood samples and stored at −70°C until analyzed. The Bio-Plex Human 8-plex kit  was used to detect IL-2, IL-4, IL-6, IL-10, IFNγ, GM-CSF and TNFα levels on Luminex200 xMAP analyzer system , according to the manufacturer's instructions.2 or Fisher's exact test. Continuous data were analyzed by using Spearman's rho correlation analysis. Mann-Whitney U test was used for data that did not have a normal distribution. Correlation among cytokine levels was analyzed by hierarchical cluster analysis. All analyses were performed by SPSS software . P value <0.05 was considered to be statistically significant.Average of data was presented as The demographic and clinical data of 571 HFMD patients are collected and summarized in 9/L (p = 0.040). Results of multivariate analysis indicated five independent risk factors (RR>26/min (p<0.001), Age<4 yo (p<0.001), GLU>8.3 mmol/L (p = 0.011), LYM>40% (p = 0.010), and ALT>40 U/L (p = 0.045)) >26/min (p<0.001), age<4 yo (p = 0.001), GLU>8.3 mmol/L (p = 0.008), CL<98 mmol/L (p = 0.026), and WBC>1.2×10 0.045)) Table 2.Using single parameter to predict disease severity is not a reliable and accurate way to assist diagnosis and initiate early intervention. Therefore, we analyzed various combinations of different important parameters we found above. We tested the positive rates of different combinations of the seven markers and calculated Popularity, Confidence ratio (CR) and Support Table 3.The most reliable combination is “GLU>8.3 and CL<98 and RR>26”, which offered 100% CR, which means all patients with this combination belonged to severe cases. The second most reliable combination is “ALT>40 and LYM>40% and RR>26 and Age<4 yo”, whose CR was 92.9%. Among the top 10 combinations, there are two very simple ones, “GLU>8.3 and CL<98” and “GLU>8.3 and RR>26”, whose CR were 87.5% and 87.1%, respectively. The combination “RR>26 and Age<4 yo” gave the best Popularity (59.3%), which came with the highest Support (42.3%) and a CR of 71.4%.To further analyze the links between risk factors and severe cases, and correlations among risk factors, we created a correlation web Figure 2Cytokines are among the key mediators of immune responses to invasion of virus and other pathogens. Therefore, the levels of IL-2, IL-4, IL-6, IL-10, IFNγ, GM-CSF, and TNFα in all enrolled subjects were tested upon admission, and results are presented in Based on our analysis, we tried to create a practical evaluation system for severe HFMD cases, since most combinations with high CR came with a very low popularity, indicating that using only CR as an indicator for severe cases was not practicable Table 3.9/L were found to be associated with severe cases. And results of multivariate analysis indicated five independent risk factors  >26/min, blood glucose (GLU)>8.3 mmol/L, age<4 yo, percentage of lymphocytes (LYM)>40%, blood chlorine (CL)<98 mmol/L, and white blood cell counts (WBC)>1.2×10>40 U/L) Table 2.In addition to single-factor analysis, we further analyzed the use of different combinations of risk factors. Based on 3 parameters , combinations of risk factors were evaluated. Combinations with the best confidence ratio should be the best indicator for severe cases by definition. Therefore, “GLU>8.3 and CL<98 and RR>26” (CR = 100%) is our top indicator, followed by “ALT>40 and LYM>40% and RR>26 and Age<4 yo” (CR = 92.9%). All combinations with a CR> = 80% came with a case number<32 and a support<4.9%, except “CL<98 and LYM>40% and RR>26 and Age<4 yo”, whose case number was 73 and support was 10.4%. On the contrary, combinations with a lower CR (70.0% to 79.9%) had a much higher case number and support in average, if compared with those with a CR>80.0%. Among combinations with a lower CR, 37.9% (11/29) had a case number>100 , and 48.3% (14/29) had a support>10%. This implies that these combinations are more commonly seen in clinical practice and should not be ignored. Although combinations with high CR may help identifying severe cases, their popularity is very low Table 3.Prior studies have demonstrated the correlation between certain cytokines and HFMD. The study on ratio of cytokine levels in CSF over in plasma had suggested the possible role of IL-8 in response to EV71 infection complicated with PEIn conclusion, five independent risk factors, along with indicative combinations of risk factors, for severe cases were identified, and a scoring system was created to facilitate the use of indicators for early medical intervention.


Age related cataract is the leading cause of blindness in the world today. The association between DNA damage to the lens epithelium and the development of lens opacities has been reported in many studies. Polymorphisms of DNA repair enzymes may affect repair efficiency and thereby lead to the development of age related cataract.XPD) codon 312 and X-ray complementing group1 (XRCC1) codon 399, in a sample of 208 cataract patients  and 151 sex and age matched healthy controls. XPD genotype was determined by Amplification Refractory Mutation System (ARMS) while XRCC1 was genotyped using the PCR-RFLP method.In this study, we aimed to determine the frequency of polymorphisms in two DNA repair enzyme genes, xeroderma pigmentosum complementation group (XPD-312 Asn/Asn genotype in cataract patients (21.6%) and healthy controls . Considering the types of cataract, XPD-312 Asn/Asn genotype was found to be significantly different in patients with cortical (29%) type in comparison to controls . No statistically significant difference was found for the genotypic and allelic distributions of the polymorphism in XRCC1. The MDR interaction analysis revealed weak synergism between the markers XPD-Asp312Asn and XRCC1-Arg399Gln contributing to cataract. It also showed that the AA genotype of XPD-Asp312Asn polymorphism when present in combination with the GA genotype of XRCC1-Arg399Gln had a fivefold and with AA had a fourfold risk for developing cataract.There was a significant difference between frequencies for XPD codon 312 may be associated with the development of maturity onset cataract. This is the first report on the association of XPD Asp312Asn polymorphism with maturity onset cataract.The present study suggests that a polymorphism in  Age related cataract is the leading cause of blindness in the world today. It is a multifactorial disease caused by the interactions between the gene and environmental factors. Various risk factors such as diabetes, female gender, sunlight or exposure to UV light, or nutritional deficiencies have been implicated in the development of cataract . EndogenAssociation between oxidative stress and DNA damage has been well known  and manyDNA repair enzymes continuously monitor chromosomes to correct damaged nucleotide residues generated by exposure to cytotoxic compounds or carcinogens . RecentlXPD) and X-ray complementing group I (XRCC1) have been studied extensively [XPD is required for excision repair of UV damaged DNA and is an important component of nucleotide excision repair (NER) whereas XRCC1 is involved in single strand breaks and base excision repair (BER). Several studies have pointed out these two genes as good biomarkers of DNA damage [To date more than 100 DNA repair genes have been identified and their polymorphisms have been reported to be related with some diseases. Among them, polymorphisms of xeroderma pigmentosum complementation group D  and XRCC1 coodon 399 (Arg-Gln), which are the most frequent and commonly studied polymorphisms of these two well known DNA repair genes.We therefore investigated, in the cataract patients the frequency of polymorphisms in The present case-control study includes a total of 208 patients with age related cataract  and 151 sex and age matched healthy controls. The patients were recruited from the Sarojini Devi Eye Hospital and Institute of Ophthalmology, Hyderabad, India. Diagnosis of different types of cataracts was done based on slit lamp examination by the ophthalmologist following LOC-III classification. The patients and controls were explained about the purpose and outcome of the study and only those who volunteered to participate in the investigations alone were considered. The study was approved by the Institutional ethical committee.Only patients with primary cataracts were included in the present study. Any patients with a secondary cataract arising due to trauma, action of toxins, inflammations, and degenerative ocular diseases were excluded. In addition, patients with associated conditions like diabetes, hypertension, myopia, glaucoma, thyroid syndromes, and cataract inducing medications (like steroids) were not considered. Controls were selected randomly from the same population and without the history of cataract, diabetes, hypertension, thyroid, and other ocular diseases.From all the patients and controls included in the study, information pertaining to sex, age, age at onset, duration of disease, type of cataract, information on habits, diet, and a detailed three generation family history was collected using a specific proforma.Venous blood samples were obtained from patients and controls in EDTA vaccutainer tubes. DNA was isolated from these samples using rapid non-enzymatic method described by Lahiri and Nurnberger .XPD genotype was determined by Amplification Refractory Mutation System (ARMS) where in a 150 bp fragment was generated using reverse primer 5′-CAG GAT CAA AGA GAC AGA CGA GCA GCG C-3′; G allele forward specific primer 5′-GTC GGG GCT CAC CCT GCA GCA CTT CGG C-3′; and A allele forward specific primer 5′-GTC GGG GCT CAC CCT GCA GCA CTT CGA T-3′. An internal control primer was included in each reaction  at a 1:5 dilution relative to allele specific primers. Genotypes were typed as GG and AA depending on the development of bands when primers specific for allele G (as GG) or allele A (as AA) were used. Samples were typed as heterozygotes (GA) when bands were seen with both the primers.XRCC1 genotype was determined by PCR RFLP wherein a 615 bp fragment was generated by primer pair forward 5′-TTG TGC TTT CTC TGT GTC CA-3′ and reverse 5′-TCC TCC AGC CTT TTC TGA TA-3′. The PCR reaction was performed in a 10 µl volume of 20 pmoles of each primer, 0.2 mM each dNTP, 1 µl buffer and 0.25 U Taq DNA polymerase, with a initial denaturation of 94 °C for 5 min, followed by 30 cycles of 30 s at 94 °C, 30 s at 61 °C and 45 s at 72 °C and finally 7 min at 72 °C. Following amplification, the PCR products were digested with 2U of Msp1 restriction enzyme and incubated at 37 °C overnight. The wild type Arg allele for codon 399 was determined by the presence of two bands of 374 and 221 bps, while the mutant Gln allele was determined by the presence of the uncut 615 bp band.SPSS (version 17) package was used to analyze the data for descriptive statistics and computation of means and standard deviations. χ2 statistics was computed for significance of differences in the distribution of genotypes between patients and controls and also between the cohorts  in the two groups. The frequencies of the marker alleles were estimated by allele counting method and tested for Hardy–Weinberg equilibrium. Odds ratios (OR) and 95% confidence interval (CI) were computed for different combinations of genotypes to estimate the risk contributing to the onset of cataracts.Multifactor Dimensionality Reduction (MDR) analysis was performed using MDR software to determine the presence of epistatic interaction and the genotypic combination of the two genes that may confer high or low-risk for cataract development. Evaluation of gene-gene and gene-environment interaction was performed using four step process outlined by Moore et al. . The anaXPD and XRCC1 in both cases and controls are shown in The genotypic and allelic distributions of the polymorphisms in XPD-Asp312Asn genotypes were consistent with Hardy–Weinberg equilibrium among the controls (p=0.995) but not among the patients (p=0.009) indicating possible association of this polymorphism with age related cataracts. It was observed that though the allele frequencies were not different between the patients and controls, there was a significant difference between the frequencies of XPD-312 Asn/Asn genotype in cataract patients (21.6%) as compared to controls  even after Boneferroni correction (p=0.06). The OR estimates revealed a possible risk of XPD-312 Asn/Asn genotype for developing cataract . Also when the three genotypes were reduced to two by treating the least common genotype as the recessive one, similar results were obtained for the polymorphism  indicating nearly twofolds risk for Asn/Asn genotype for developing cataracts.The distribution of XRCC1-Arg399Gln (G>A) polymorphism, the genotypes were in accordance with Hardy–Weinberg equilibrium among the cases (p=0.083) and controls (p=0.207). No statistically significant difference was found for the genotypic and allelic distributions of the XRCC1 polymorphism between the patients and controls.Considering the XPD-Asp312Asn and XRCC1-Arg399Gln polymorphisms in controls and cataract types.XPD-Asp312Asn polymorphism did not differ significantly in the cataract types. However, investigation of the genotype frequencies in different cataract types and controls revealed a significant difference between frequencies for XPD-312 Asn/Asn genotype in patients with CC (29%) and controls (13.2%). The OR estimates revealed that XPD-312 Asn/Asn genotype had more than twofolds risk for developing CC  even after Boneferroni correction (0.06).Compared with the controls, the allele frequencies of XRCC1 gene between controls and cataract types.No statistically significant difference was found for the genotypic and allelic distributions of the polymorphism in XPD Asp312Asn when present in combination with GA genotype of XRCC1 showed a fivefold risk (23/4) while when present with AA a fourfold risk (4/1) for developing cataract.The present data included polymorphisms Asp312Asn Arg399Gln as the over all best model with maximum testing accuracy of 0.5100 and CV consistency of 10/10. But the combination was not found to be significantly contributing to cataract. High-risk (dark gray) and low-risk (light gray) genotypic combinations were determined based on the threshold value for the present data which was 1.37 208/151; . It was XPD-Asp312Asn and XRCC1-Arg399Gln contributing to cataract.Successful repair of damaged DNA relies on the coordinated action of many repair enzyme systems. Age dependent decline or imbalance of the activities of the DNA repair enzymes will result in the compromise of the overall capacity of repair for the damaged DNA molecules. This may account for the ever-increasing accumulation of oxidative damage and mutation to DNA in the aging tissues ,24.Age-related changes occur also in the human lens. The human lens consists of three metabolically different zones: the epithelium, the cortex and lens nucleus or core . The lenXRCC1 is a DNA repair gene that is emerging as an essential element in the repair of both damaged bases and SSBs. On the other hand, the XPD gene is required for excision repair of UV-damaged DNA and is an important component of NER. Several studies have linked these two polymorphisms with biomarkers of DNA damage [Common polymorphisms in DNA repair enzymes have been hypothesized to result in reduced capability to repair DNA damage ,32. XRCCA damage ,21.XPD 312 Asn/Asn genotype for developing cataract, especially cortical cataract. But there was no significant association between XRCC1 polymorphism and cataract development. Interaction analysis also revealed greater risk for AA genotype of XPD Asp312Asn polymorphism in combination with AA or GA genotype of XRCC1 polymorphism (To our knowledge, the present study is the first report from Indian population on the association of DNA repair enzymes with age related cataract. We found a possible risk for morphism .XPD codon 751 may be associated with the development of maturity onset cataract. Thus SNPs in XPD gene may be expected to have a role in the development of an UV-related disease, like maturity onset cataract. In our study, we found a possible risk of XPD-312Asn/Asn genotype in the development of cataract, especially against the development of cortical type cataract. This is the first report on the association of XPD- Asp312Asn polymorphism with maturity onset cataract. However, further studies are required to understand the precise mechanisms by which genetic polymorphisms of DNA repair genes influence the process of lens opacification.Previous studies have reported an association between the UV light exposure and the development of cortical cataract. A review of 22 epidemiologic studies by McCarty and Taylor , reveale


The associations between the polymorphisms of interleukin-8 (IL-8) gene and gastric carcinogenesis have been extensively investigated in recent years. However, the results remain conflicting rather than conclusive.A meta-analysis of 18 eligible studies was performed to evaluate the association of IL-8 -251A > T polymorphism with risk of gastric carcinogenesis. A systematic literature search of MEDLINE, Embase, and Web of Science, CNKI databases was conducted. Statistical analysis was performed by using the Revman 5.1 software and the Stata 12.0 software.P = 0.02), especially gastric cancer , but not associated with the risk of precancerous lesion . Analysis stratified by ethnicity may seem that IL-8 -251A > T polymorphism was susceptible to gastric cancer in Asian population, but not in Caucasian population.Of the 293 unique studies identified using our search criteria, 18 studies fulfilled our inclusion criteria and were included in the meta-analysis. These studies cumulatively reported 5,321 cases and 6,465 controls. The combined results based on all studies showed that the IL-8 -251A > T polymorphism was associated with the risk of gastric carciongenesis (A vs. T: OR: 1.14 , Our meta-analysis results provide evidence that IL-8 -251A > T polymorphism is significantly associated with increased risk of gastric carcinogenesis in Asian population, particularly in gastric cancer. Further large and well-designed studies are required to confirm this conclusion. Helicobacter pylori infection, life style, socioeconomic status, and environmental factors [Although the incidence of gastric cancer has recently declined in several countries, it is still a serious health problem and remains the world’s fourth common malignancy and the second leading cause of cancer death -3. Curre factors . In addi factors , althougChemotactic cytokines, produced by tumor and endothelial cells, could play an important role in cancer, such as increasing angiogenesis, stimulating tumor progression, enhancing tumor cell migration, and facilitating evasion of immune surveillance ,7. As a IL-8 gene, which is located on chromosome 4q12-21, contains four exons and three introns, and exhibits functional polymorphisms, fifteen of which have been characterized . Among tOf the 293 unique studies identified using our search criteria, 18 case–control studies fulfilled our inclusion criteria and were included in the meta-analysis -39. ThesThe main features of the studies included in the meta-analysis were shown in Table Table P = 0.02) , and homozygous comparison (AA versus TT) , respectively, but not in the dominant model (AA vs AT + TT)  and the heterozygous comparison (AT versus TT)  (Table P < 0.05), dominant model , recessive model , homozygous comparison , and heterozygous comparison . However, no significant association between this polymorphisms and gastric carcinogenesis risk was observed in all comparison models in Caucasians population  , whereas no significant risk was observed under dominant model . Moreover, we found that gastric cancer risk was significant increased in Asian population under allele comparison , dominant model , recessive model , homozygous comparison , and heterozygous comparison . However, no significant association between this polymorphisms and gastric cancer risk was observed in all comparison models in Caucasians population , dominant model , recessive model , homozygous comparison , and heterozygous comparison.The information on the association between IL-8-251 A > T polymorphism and gastric precancerous lesions was available from four articles evaluated in this study Figure . No remaP = 0.484) , which indicated low risk of publication bias in this meta-analysis.Begger’s funnel plot and Egger’s linear regression test were performed to assess the publication bias of included studies. The shapes of the funnel plots seemed symmetrical in the allele comparison model  Figure . Egger’sThe pathogenesis of gastric carcinogenesis involves environmental factors, molecular signaling pathways, and host genetic factors . The rolP = 0.07) and the recessive model (AA + AT vs. TT) (P = 0.02) both showed increased (1.18 fold) risk of gastric carcinogenesis. Even though the precise role of IL-8 -251A > T polymorphism in the development of gastric carcinogenesis is unknown, a plausible mechanism is that the mutations of IL-8 gene might increase gene transcription after binding to its high affinity cell surface receptor, which eventually attribute to the correlation between IL-8 and gastric carcinogenesis risk.The present meta-analysis summarized the correlation between the IL-8-251 A > T polymorphism and susceptibility to gastric carcinogenesis in 18 studies. The results suggested that the AA and AT genotypes of IL-8 -251A > T polymorphism appears to be associated with an overall increased risk of gastric carcinogenesis and is discovered as a risk factor of gastric cancer. Subgroup analysis by ethnicity allowed us to look for potential ethnic differences in the association. In the Asian population, the A-allele was associated with increased risk of gastric carcinogenesis and gastric cancer based on allele comparison, dominant model, recessive model, homozygote comparison, and heterozygous comparison. However, for the Caucasian population, IL-8 -251A > T polymorphism was not associated with increased risk of gastric carcinogenesis and gastric cancer. The A allele seemed to be dominant, but in the precancerous lesion subgroup, no significance was noted for all models. The possible reason could be that genetic risk factors differ between gastric cancer and precancerous lesions.The IL-8 promoter is estimated to be 1,500 bp. Several reports have shown relationship between IL-8 gene polymorphisms and human diseases, and all of them have focused on the A/T polymorphism at -251 upstream from the transcriptional start site. IL-8 -251A > T polymorphism has been associated with altered transcription levels of IL-8 by regulating the transcriptional activity of the gene and then proved to affect susceptibility to a large number of diseases. In the present analysis, we found an overall increase in gastric carcinogenesis of one or two allele variants as compared to wild allele T and homozygous TT genotype. After stratification into dominant and recessive genetic models, the dominant model (AA vs. AT + TT) , Embase , and web of Science , and CNKI  databases was conducted by two study investigators (D.C. and Y. H.) independently for all relevant articles. Key words used in the research included “Interleukin-8”, “IL-8”, “CXCL8”, “gastric cancer”, “stomach cancer”, “precancerous lesion”, “polymorphism”, “SNP”, “gene variant”, “gene mutation”, and “gastric tumor”.Studies eligible for inclusion in this meta-analysis should meet the following criteria: (a) case–control studies or cohort studies focused on association between IL-8-251 A > T polymorphism and risk of gastric carcinogenesis; (b) patients have pathologically or histological confirmed gastric cancer and/or precancerous lesions; (c) The studies provided the number of cases and controls for various genotypes. The exclusion criteria of the meta-analysis were: (a) animal studies; (b) meta-analyses, letters, reviews or editorial comments; (c) studies with duplicate data or incomplete date. When an individual author published several articles obtained from the same patient population, only the newest or most complete article was included in the analysis.P value for HWE. An attempt was made to contact authors if data presentation was incomplete or if it was necessary to resolve an apparent conflict or inconsistency in the article. Any disagreements were resolved by consensus.Information was carefully extracted from all the eligible publications. The following data were collected from each study: first author’s name, publication date, country, ethnicity, source of controls, genotyping method, total numbers of cases and controls, number of cases and controls for each IL-8-251 A > T polymorphism, and P value of heterogeneity test was <0.1 (or I2 > 50%). The odds ratio (OR) and 95% confidence interval (CI) were calculated for each study, and the combined OR and 95% CI were calculated for all eligible studies. OR was the proportion of the exposed population in whom disease has developed over the proportion of the unexposed population in whom disease has developed in a case–control study. The significance of the combined OR was determined by the Z-test, in which P < 0.05 was considered significant. Heterogeneity assumption was assessed by the chisquare based Q test and was regarded to be statistically significant if P < 0.10. The potential publication bias was assessed by Begg’s funnel plot and Egger’s test [Review manager 5.1 program provided by the Cochrane Library and Stata  were used to perform all the statistical analysis. The association was evaluated with the use of the allelic comparison (A versus T), as well as the dominant model (AA versus AT + TT), the recessive model (AA + AT versus TT), the homozygous comparison (AA versus TT), and heterozygous comparison (AT versus TT), respectively. Two models of pooling data for dichotomous outcomes were conducted: the random-effects model and the fixed-effects model. The pooled statistical analysis was calculated using the fixed effects model, but a random-effect model was performed when the r’s test ,43.IL-8: Interleukin-8; OR: Odds ratio.The authors declare that they have no competing interests.DC conceived and designed the study. DC and YH drafted the manuscript. WZ and YM collected the data. DC performed the data analysis. All authors have read and approved the final manuscript.


Primary or adult type hypolactasia, the most common enzyme deficiency in the world, is due to reduced lactase activity in the intestinal cell after weaning. Lactase non-persistence is inherited as an autosomal recessive trait. A DNA variant, single nucleotide polymorphism C/T−13910 which is located on 13910 base pairs (bp) upstream of the lactase gene (LCT) at chromosome 2 has been show to associate with the lactase persistence/non-persistence. The prevalence of the C/T-13910 variant is different for hypolactasia in European, Asian, African-American and Northern African populations. In this study, we investigated, for the first time the allele frequent of the single nucleotide polymorphism C/T−13910 in the Iranian population in khorasan province with hypolactasia.P-value of <0.05 was considered statistically significant.Peripheral blood was collected from 100 subjecs with primary hypolactasia and 100 healthy individuals as a control group. Genomic DNA was extracted. The genotype was analyzed with the PCR-RFLP method. A statistical analysis was performed by chi-square test using SPSS software. A P=0.03), and in genotype frequency between case and control groups (P=0.006).In case group allelic frequency for SNP T-13910C  was respectively 95%, 5% vs. control group 86% and 14%. Genotype frequency  in patient group was 90%, 10%, 0% vs. control group 74%, 24% and 2%. So according to our findings, there were significant differences between allelic frequencies (Based on our results, analysis of C\T-13910 polymorphism can be used as a simple genetic test for diagnosis of primary type hypolactasia in the Iranian population. Lactose disaccharide is the main carbohydrate in mammalian milk that is comprised of glucose and a galactose molecule. Lactase or lactase-phlorizin hydrolase is a brush border membrane enzyme in the intestinal epithelial cells that catalyzes lactose , 2. PrimDuring childhood, lactase activity reduces to about 10-15% of its activity at birth. This condition is genetically programmed and irreversible reduction of lactase occurs in more than 75% of the world’s population -6.et al (1988) showed that the LCT gene is located on chromosome 2  analyzed the region flanking the LCT gene at 2q21. They found that C/T polymorphismtat which is located on 13,910 base pairs upstream of the LCT gene, in intron 13 of the MCM6 gene; have complete association with lactase persistence/non-persistence trait  and 100 healthy individuals as a control group . Informed consents were obtained from all participants. The exclusion criteria were disease associated with secondary hypolactasia such as celiac, severe gastroenteritis, crohns disease, and ulcerative colitis.Genetic testGenomic DNA was extracted from leukocytes collected in EDTA tubes . The previously described primers, LAC-C-M-U: 5′-GCTGGCAATACAGATAAGATAATGGA-3′ (position 26611–26636) and LAC-C-L-2: 5′-CTGCTTTGGTTGAAGCGAAGAT-3′  was used to amplify the region flanking the C/T13910 polymorphism (12). Hundred ng of genomic DNA was used as template in a reaction volume of 50 μL, containing 36.4 μl ddH2O, 5 μl 10X reaction buffer, 25 mM MgCl2, and 10 mM dNTP, 10 pmol of each primer and 5U of Taq DNA polymerase (Genet Bio). Cycling conditions were 94°C for 5 min, followed by 30 cycles of 40 sec at 94°C, 40 sec at 58°C and 40 sec at 72°C, and 5 min at 72°C as the final extension step. Mulcare et al (13) introduced a base change in the penultimate base of the LAC-C-M-U primer (G instead of T) so that the PCR product could be digested by HinfI as the T allele exists. PCR products (10 μl) were digested with restriction endonuclease HinfI (0.25 U) incubated at 37°C overnight , followed by enzyme deactivation at 65°C for 20 min. HinfI digestion of PCR products with the T allele resulted in 177 bp and 24 bp fragments; digestion of the C allele generated a single fragment of 201 bp.Positive controls were included to confirm complete digestion. Digested PCR products were observed on a 3% agarose gel stained by ethidium bromide. Samples showing a single band of 201 bp were typed as the CC genotype and a single band of 177 bp as the TT genotype; two bands of 201 bp and 177 bp represented the CT genotype . The CT Statistical analysisStatistical analysis was performed by chi-square test using SPSS software. A P- value of <0.05 was considered statistically significant.For case and control groups, tests of the validity of Hardy-Weinberg equilibrium showed no deviation from expectation. In the case group, allelic frequency for SNP T-13910C  was respectively 95% and 5% vs. control group 86%, 14% in genotype frequency  in patient group was 90%, 10%, 0% vs. control group 74%, 24% and 2% displayed in P= 0.03), and in genotype frequency between case and control groups (P= 0.006).Thus, according to our findings there were significant differences between allelic frequencies . There was significant difference in CC genotype between patient and control groups (P= 0.006). Based on our results, analysis of C\T-13910 polymorphism can be used as a simple genetic test and a non-invasive test for diagnosis of primary adult type hypolactasia. In this study we showed for the first time that genetic test could be used with high sensitivity, time and cost effectiveness for hypolactasia in Iran.Due to this condition, frequency of recessive allele, C-13910 allele, becomes very high in comparison with dominant allele. However, it is possible that there are additional mutations that have not been discovered yet. However, frequency of C allele that is associated with lactose intolerance was significantly higher in patient group compared with control group (In this work we showed for the first time that, there is association between CT-13910 polymorphism and hypolactasia in the Khorasan population. Analysis of the C/T-13910 variant can be a very specific and sensitive test for predicting hypolactasia in a patient population with suspected lactose malabsorption.


CLPTM1L), have been implicated in susceptibility to lung cancer through genome-wide association studies (GWAS); however, subsequent replication studies yielded controversial results.Common genetic polymorphisms on chromosome 5p15.33, including rs401681 in cleft lip and palate transmembrane 1-like gene  group conferred an OR of 0.801 (95% CI = 0.654–0.981) under the dominant model. The meta-analysis comprising 9111 cases and 11424 controls further confirmed the significant association in the dominant model . By stratified analysis, we revealed that ethnicity and study design might constitute the source of between-study heterogeneity. Besides, the sensitivity and cumulative analyses indicated the high stability of the results.The results from our case-control study and meta-analysis provide convincing evidence that rs401681 is significantly associated with lung cancer risk. Lung cancer is the most common cancer throughout the world. Globally, it accounts for 13% (1.6 million) of the total cases and 18% (1.4 million) of the deaths in 2008 CLPTM1L  Ptrend = 0.259) P = 1.1×10−5) Ptrend = 1.89×10−4), thereafter, neither Bae et al. Recent genome-wide association studies (GWAS) have implicated multiple novel single nucleotide polymorphisms (SNPs) to lung cancer susceptibility TERT locus polymorphisms and risk of different cancers in a time span of 2003–2011, has presented a modest risk reduction  of rs401681 for lung cancer As above, the outcomes remain ambiguous and conflicting, which is probably due to the modest effect of this SNP, leading to the lack of power in small genetic association studies. It is also potentially owning to the so-called “winner’s curse” phenomenon that OR of disease variant is usually overestimated in the first positive study. According to the reported OR of this study, the necessary sample size of replication studies would be underestimated, then the underpowered replication would be difficult to succeed In the present study, a total of 611 lung cancer cases and 1062 cancer-free controls were recruited from Tongji Hospital of Huazhong University of Science and Technology (HUST) between 2009 and 2011. All subjects were unrelated ethnic Han Chinese in Wuhan region. Cases were histopathologically confirmed with all lung cancer types and have not received any treatment prior to blood samples collection. Controls were selected randomly from a physical examination programs at the same hospital in the same time period as the patients were enrolled. The case patients and control subjects are adequately matched in terms of gender and age (±5 years). At recruitment, 5-ml peripheral venous blood was collected from each subject after informed consent was obtained. This study was approved by ethnics committee of Tongji Hospital of Huazhong University of Science and Technology.Genomic DNA was extracted from the peripheral blood sample applying the RelaxGene Blood System DP319-02  in accordance with the manufacturer’s instructions. The rs401681 was genotyped with the TaqMan SNP Genotyping Assay  on a 7900HT Fast Real-Time PCR System . 5% duplicated samples were randomly selected to assess the reproducibility for quality control, with a concordance rate of 100%.2χ test and t test were applied to estimated differences in demographic variables and distributions of genotypes between cases and controls. Hardy-Weinberg equilibrium (HWE) was calculated using goodness-of-fit 2χ test for genotypes in controls and a value of P<0.05 was considered as significant disequilibrium. After adjusting for age, sex and smoking status, unconditional multivariate logistic regression was employed to estimate genotypic odds ratio (OR) and its 95% confidence interval (95% CI), with the reference of the common homozygote. In order to avoid the assumption of genetic models, dominant and additive models were also analyzed. All above statistical analyses were performed with the SPSS 20.0 software.The rs401681, CLPTM1L, TERT, or 5p15.33′ combined with ‘lung cancer, NSCLC’ without language restrictions. Basing on the above searching strategy, we further searched in Chinese Biomedical (CBM) database. Meanwhile, references listed in the retrieved articles were scanned. Reviews and comments were also checked for additional studies. Articles which met the following criteria were included: (1) case-control or nested case-control study assessing the association between rs401681 and lung cancer risk; (2) providing data for calculating genotypic odds ratio (ORs) with corresponding 95% confidence interval (95% CI); (3) genotypes in controls being in Hardy-Weinberg equilibrium (P>0.05) (4) studies of humans. Whenever studies pertained to overlapping subjects, the one with larger sample size was selected to avoid duplication.A systematic literature searching on PubMed, EMBASE, and ISI Web of Science databases up to April 2012 was performed, using the keywords ‘2-χbased Cochran’s Q statistic and the 2I metric to assess between-study heterogeneity. Heterogeneity was considered significant at P<0.10. The subsequent cut-off points of the quantity 2I were utilized to quantify heterogeneity: 2I = 0−30%, no or marginal between-study heterogeneity; 2 = I30%–75%, mild heterogeneity; 2I = 75−100%, notable heterogeneity P for Q statistic greater than 0.1, the fixed-effects model (Mantel-Haenszel method) was adopted to compute the pooled ORs and 95% CIs P values are two-tailed with a significant level at 0.05 except for Q test for heterogeneity.The following information from each study was extracted: first author’s last name, year of publication, geographic location, ethnicity of study population, study design, genotyping method, numbers of cases and controls, frequencies of genotypes in cases and controls. Pooled frequency of the T allele in different ethnicities and histological types was estimated by the inverse variance method previously used by Thakkinstian et al http://compbio.cs.queensu.ca/F-SNP/), “FastSNP” (http://fastsnp.ibms.sinica.edu.tw/pages/input_CandidateGeneSearch.jsp) and “SNP Info” (http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm).To predict the function of rs401681, bioinformatics analyses were conducted with three online bioinformatics tools“F-SNP” (P = 0.475) and age (P = 0.287) distribution. Males were 68.6% among cases compared with 70.2% among controls and the median age was 61.5 for cases and 61.0 for controls. As expected, more smokers were presented in the cases than in the controls , considering that cigarette smoking is the major etiological factor for lung cancer. Herein, smokers were defined as those who had smoked at least one cigarette per day for 12 months or longer at any time in their life, while non-smokers were defined as those who had not. Of the cases, 427 (69.9%) were histopathologically confirmed as NSCLC, the most common type that accounting for approximately 85% of lung cancer in general, including adenocarcinomas, squamous cell carcinomas and large cell lung carcinomas.A total of 611 incident cases of overall lung cancer and 1062 frequency-matched controls were enrolled in this study. As shown in P = 0.073). Genotypes in controls complied with Hardy-Weinberg equilibrium (P = 0.932). In the multivariate logistic regression model adjusted for age, sex and smoking status, individuals with the CT genotype had a significant decreased risk of lung cancer  compared to those with the CC homozygote. A dominant model was performed to increase statistical power, by combining the TT with the CT into a T carrier (TT plus CT) group. And the result showed that the T carriers also present significantly reduced risk , suggesting a dominant effect of this polymorphism on cancer in Chinese population. Likewise, significant associations were found in the additive models, with per-T-allele OR of 0.856 (95%CI = 0.734–0.998). To explore the variant’s effect on NSCLC, we also compared the genotype distribution between the NSCLC cases and controls. Similar positive association and stronger genetic effect were found in this subtype of overall lung cancer .The distribution of rs401681 genotypes among subjects are displayed in However, when we further examined with stratifications by smoking status (smokers and non-smokers) and the median age (age≤61.0 and age>61.0), no valid associations were detected under the dominant model except in the group who were under the age of 61.0. Null association might be due to the small number of subjects after stratification in the current study. The significant finding of the younger group is in line with the conception that genetic susceptibility is often associated with an early age of disease onset. But when we particularly defined the early-onset cases as who were ≤50.0 years of age and performed stratification analysis, we observed no significant associations .As shown in P for heterogeneity>0.10), pooled T allele frequencies were 44.6% (95CI = 43.4%–45.9%) in Caucasian controls and 32.0% (95%CI = 31.2%–32.7%) in Asian controls, which were similar to those reported in HapMap database of 43.4%, 31.0% and 35.8% for Caucasians, Chinese and Japanese respectively.Under fixed model (either P for heterogeneity<0.001). Under random-effects model, the pooled frequency of the T allele was 36.9% (95%CI = 26.2%–47.5%) in the NSCLC cases of our whole study, which was slightly higher than that of 31.6% (95%CI = 26.3%–36.8%) in overall lung cancer cases.Significant between-study heterogeneity were observed in both subunits of NSCLC and lung cancer (P for heterogeneity<0.10), and its OR which was pooled under random-effects model are 0.778 (95%CI = 0.674–0.900). On the other hand, fixed-effects model was applied for heterozygous, dominant (P for heterogeneity>0.10). All of these models conferred significant decreased risk of lung cancer, with ORs of 0.864 (95%CI = 0.813–0.917), 0.842 (95%CI = 0.795–0.891), 0.871 (95%CI = 0.835–0.908), respectively  in Caucasian. In Asian population, decreased risk was also conferred without evidence of heterogeneity in all genetic models except for the TT genotypic model . The mildly reduced risk from one copy of T allele to two copies in Chinese  when the study of Yoon et al. was excluded , with the same null association .Since significant between-study heterogeneity for the Asian studies was observed in the TT genotypic model, we also performed a sensitivity analysis among those studies. And we have found that the heterogeneity reduced most  and CLPTM1L . Although little was known about the function of rs401681 which was situated within the intronic region of CLPTM1L, our bioinformatics analysis indicated that it might be transcription regulatory and further affect the expression of the gene. CLPTM1L, named for its homology with Cleft Lip and Palate Transmembrane Protein 1 that disrupted in a family with cleft lip and palate CLPTM1L mRNA was discovered in many kinds of cancer CLPTM1L and its role in tumorigenesis is thus far unknown. But a recent study reported that CLPTM1L was a commonly overexpressed anti-apoptotic factor in lung cancer, suggested it a inhibitor role in genotoxic stress induced apoptosis, and therefore identified CLPTM1L as an important factor influencing survival of DNA damaged tumor cells and potentially lung cancer susceptibility These findings are biological plausible to some degree. rs401681 is located at 5p15.33, a susceptibility region for lung cancer encompassing two known genes CLPTM1L might be relevant not only in the light of its own biological activity but also because it is in LD with TERT. The entire gene CLPTM1L resides in a 62-kb region of high linkage disequilibrium (LD) that encompasses the 5′-end of TERT, its promoter TERT is the reverse transcriptase component of telomerase CLPTM1L is in linkage disequilibrium with some causal locus in the promoter of TERT which are hitherto uncharacterized.The results of our case-control study indicated that the minor allele T of rs401681 was significantly associated with protective effect to lung cancer in heterozygous, dominant and additive genetic models, but not in the homozygous possibly because of our relative small sample size. Despite similar significant relationship maintained in our meta-analysis in all genetic models, obvious between-study heterogeneity can not be ignored in the homozygous model. A comprehensive stratified analysis was conducted to interrogate the potential source of heterogeneity. After stratified by ethnicity, all genetic models of the T variant allele were significantly associated with reduced cancer risk with disappeared heterogeneity in Caucasians, while in Asians, all genetic models conferred decreased risk without significant heterogeneity except the homozygous model. The apparent difference between the two homozygous ORs implied different allele frequencies between Asians and Caucasians, which is supported by our study of pooled T allele frequencies and HapMap data as shown above. In view of the differences in allele frequencies and possible distinction of linkage disequilibrium patterns among populations, distinct genetic mechanisms with different genetic effect sizes for TT genotype between two populations may occur. As to the heterogeneity in Asian studies, suggested by the subgroup sensitivity analysis (data not shown), it may largely attribute to the study of Yoon et al with various kinds of genotyping methods, which are Affymetrix, TaqMan and Invader. Regarding study design, obvious heterogeneity disappeared in GWAS but still showed in replication studies, which could be also explained by the diverse genotyping methods of the replications. Because NSCLC accounts for the great mass of overall lung cancer cases to almost 85% as mentioned above, we pooled these two types of studies to enlarge sample size without concern to the potential bias of strengthening or weakening the real effect of the variant to a great extent. There weren’t much change of the heterogeneity in the subgroup of NSCLC or lung cancer overall, hinting that histological type was not likely the source of heterogeneity. At the same time, a more pronounced risk reduction for NSCLC suggested that the rs401681 variant might have larger effect on NSCLC, supporting the same situation observed in case-control study. The variance of the genotypic effect between these two subgroups could be partly ascribed to different histological type characterized by allele frequency difference, with pooled T allele frequencies of 36.9% in NSCLC cases and of 31.6% in overall lung cancer. In the whole, ethnicity and study design probably accounted for the heterogeneity of the meta-analysis.Despite the clear strength of the current comprehensive analysis strategy , several limitations should be pointed out. Firstly, the sample size of our case-control study was relatively small. Secondly, lung cancer is a complex trait related to environmental and genetic risk factors. However, insufficient environmental information limited us to further investigate the gene-environment interaction. Thirdly, lacking of functional experiments, whether this SNP is causal remained uncertain.In conclusion, our replication study in a Chinese population and the subsequent meta-analysis collectively confirm genetic involvement of rs401681 polymorphism in lung cancer susceptibility, and suggested that the variant may yield stronger effect on NSCLC. However, fine-mapping of 5p15.33 region and functional experiments is warranted to identify causal variant.Figure S1Follow chart of study selection.(TIF)Click here for additional data file.Table S1The association between rs401681 and risk of lung cancer by smoking status and age range.(DOC)Click here for additional data file.Checklist S1PRISMA checklist.(DOC)Click here for additional data file.


In this study, we evaluated the association between these polymorphisms and gallstone disease using meta-analysis and compared the hepatic ABCG5/G8 mRNA expression and biliary lipids composition in patients with different genotypes of T400K and Y54C.Data were analyzed using the Stata/SE 11.0 software and a random- effects model was applied irrespective of between-study heterogeneity. Hepatic mRNA expression of ABCG5/G8 genes in 182 patients with gallstone disease and 35 gallstone-free patients who underwent cholecystectomy were determined using real-time PCR. Genotypes of Y54C and T400K in the ABCG8 gene were determined by allelic discrimination using either genomic DNA or hepatic cDNA as template by Taqman assays. Biliary compostion in gallbladder bile was assayed in these patients as well.I2 statistics indicated no significant between-study heterogeneity for all genetic models for any of the three polymorphisms. Funnel plot and Egger’s test suggested the absence of publication bias as well. However, no association of T400K and Y54C polymorphism with hepatic ABCG8/G5 mRNA expression or biliary lipids composition was found.Ten papers including 13 cohorts were included for the final analysis. In the genotype model, the overall association between genotype with gallstone was significant for D19H , and for Y54C , or T400K . In allele model, minor alleles of D19H polymorphism  and of T400K polymorphism  were related with an increased risk of gallstone disease. However, minor allele of Y54C polymorphism  was not related with gallstone disease. Our study showed strong association of D19H polymorphism with gallstone disease. T400K and Y54C polymorphism, though to a less extent, may also relate with gallstone disease. Cholesterol gallstone disease is common in western countries The classical pathogenesis model of cholesterol gallstone formation included three defects: supersaturation of biliary cholesterol, imbalance of pro/anti-nucleation factors and impaired gallbladder motility The aims of our study are: 1) to evaluate the association between polymorphisms at D19H, T400K and Y54C and gallstone disease using meta-analysis; 2) to compare the hepatic ABCG5/G8 mRNA expression and biliary lipids composition in patients with different genotypes of T400K and Y54C.http://www.ncbi.nlm.nih. gov/pubmed/), Embase (http://www.embase.com), ISI Wed of Knowledge (http://isiknowledge.com), Wanfang (http://www.wanfangdata.com.cn) and China Biological Medicine (CBM) (http://cbm.imicams.ac.cn) with the last update as of September 2013. The keywords used for search were ‘gallstone disease’ and ‘ATP binding cassette G8 or ABCG8’ combined with ‘gene or variants or polymorphism or alleles’, all of which were MeSH terms . Only studies published in English or Chinese were identified. Afterwards, the full text of the retrieved articles were scrutinized to be sure that data of interest were included. If two or more studies shared the same studied populations, the one with the small size was abandoned. If more than one geographical or ethnic population were included in one paper, each ethnical population was considered separated.Publication were searched via public database PubMed  genotype/allele counts of ABCG8 polymorphisms between cases and controls for estimating odds ratio (OR) and 95% confidence interval (95%CI); 4) study population were adults.Gallstone disease was diagnosed by ultrasonography or operation. In some studies, cholesterol contents in gallstone was analyzed Two authors (ZY Jiang and QC) independently drew the following information from all qualified studies : first a2, female% = 65.0%). All samples were stored in −20°C until laboratory test. Patients with hepatic, renal or endocrine disorders were not included in the study. The gallstones were classified as cholesterol type by cut-face appearance or chemical analysis when necessary as reported The study protocol was approved by Ethical Committee at Ruijin Hospital and written informed consent was obtained from each participants. Three panels of patients who subjected to cholecystectomy were included in the analysis. Liver biopsies about 0.5g were taken from the right lobe of liver during the operation and snap-frozen in liquid nitrogen. Part of the samples had been used in our previous studies Hepatic total RNA was extracted with Trizol reagent  and transcribed into cDNA . Real-time quantitative PCR assays were performed using Sybr-Green . Primers (primer sequences are available on request) were designed using Primer Express 2.0 and all crossing exon-exon boundaries. Data were expressed in arbitrary units, and were normalized by the signals obtained in the same cDNA for Cyclophilin A.Single nucleotide polymorphism (SNP) analysis of the polymorphic sites Y54C and T400K in the ABCG8 gene were determined by allelic discrimination using either genomic DNA as template by commercially available Taqman assays  or hepatic cDNA as template by Taqman assay developed previously Biliary cholesterol, total bile acids and phospholipids in gallbladder bile were measured as previously described 2I statistic, which was documented for the percentage of the observed between-study variability due to heterogeneity rather than chance with the ranges of 0 to 100%  The associations between genotypes/alleles of Apo E polymorphism with GSD were evaluated by using the software Stata/SE 11.0. In the meta-analysis, we used the random-effects model with the method of DerSimonian & Laird to bring the individual effect-size estimates together. The estimate of heterogeneity was taken from the Mantel-Haenszel model We identified 17 papers potentially relevant for our study. Seven papers were excluded and 10 papers including 13 cohorts were included for the final analysis. A diagram schematizing the selection process of identified studies is presented in In the genotype model, the overall association between genotype with gallstone was significant for D19H  and DD genotypes vs DH/HH genotypes (Pegger’s test = 0.937), egger’s test = 0.511), KK/TK genotype vs TT genotype (Pegger’s test = 0.815), or Y54C Y allele vs C allele (Pegger’s test = 0.274), YY/YC genotype vs CC genotype (Pegger’s test = 0.645).Next, we used Funnel plot and Egger’s test to assess the possibility of publication bias. The resultant symmetrical funnel shape was consistent with an absence of publication bias in the funnel plot for comparison of D19H D allele vs H allele . A: Asian and Western population. B: Chinese and non-Chinese population.(TIF)Click here for additional data file.Figure S2Subgroup random-effect-based odds ratio of gallstone disease for D19H polymorphism (genotypic model). A: Asian and Western population. B: Chinese and non-Chinese population.(TIF)Click here for additional data file.Figure S3Subgroup random-effect-based odds ratio of gallstone disease for D19H polymorphism (population vs patient based controls). A: Genotypic model. B: Allelic model.(TIF)Click here for additional data file.Checklist S1Prisma checklist.(DOC)Click here for additional data file.


AGER and its circulating forms associated with the risk of CAD via a meta-analysis.Considerable efforts have been devoted to evaluating the association of the receptor for advanced glycation end-products (gene AGER and protein: RAGE) genetic variants to coronary artery disease (CAD); the results, however, are often irreproducible. To generate more information, we sought to explore four common polymorphisms of I2 = 6.7%) compared with that of overall estimate of 1.15-fold . Circulating sRAGE levels were non-significantly lower in CAD patients than in controls, whereas this reduction was totally and significantly reversed in CAD patients with diabetes mellitus . Circulating esRAGE levels were remarkably lower in CAD patients, as well as in subgroups with or without diabetes mellitus and without renal disease.Articles were identified by searching PubMed, EMBASE, Wanfang and CNKI databases before March 2013. Qualified articles had case-control designs and investigated AGER four polymorphisms  or circulating soluble RAGE (sRAGE) or endogenous secretory RAGE (esRAGE) levels associated with CAD. Twenty-seven articles involving 39 independent groups fulfilled the predefined criteria. Overall, no significance was observed for all examined polymorphisms under allelic and dominant models. When restricting groups to CAD patients with diabetes mellitus or renal disease, deviations of risk estimates from the unity were stronger than overall estimates for all polymorphisms except for G1704A due to limited available studies. For example, under dominant model, having -429C allele increased the odds of developing CAD in diabetic patients by 1.22-fold  0.99–1.51; P = 0.06; AGER genetic polymorphisms with CAD was potentiated in patients with diabetes mellitus or renal disease. Practically, circulating esRAGE might be a powerful negative predictor for the development of CAD.Our findings demonstrated that association of  AGER and protein: RAGE), which is a multiligand receptor, belonging to the immunoglobulin superfamily of cell surface molecules. The activation of AGER can evoke a wide range of signaling pathways that trigger inflammation, atherogenesis and vasoconstriction leading to coronary dysfunction, atherosclerosis and thrombosis Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Family studies suggest a strong genetic background: men with 2 or more affected parents or siblings relative to men without family history have a 3.4-fold increased risk of developing myocardial infarction AGER is mapped on chromosome 6p21.3 and spans 3.27 kb with 11 exons. The genomic sequence of AGER gene is highly polymorphic with many alleles that exhibit different functional properties and heterogeneous distributions across populations AGER genetic defects in the development of CAD; the results, however, are not often reproducible. As a caveat, failure to replicate might be attributable to the ethnicity-specific genetic profiles, the individual underpowered studies, and the lack of consideration for confounders. To generate more information, we sought to assess the association of four common polymorphisms  of AGER and its circulating forms with CAD via a meta-analysis of individual participant data from qualified case-controls studies, while addressing between-study heterogeneity and publication bias. Selection of these four polymorphisms is relatively straightforward if three or more unduplicated studies are available for a certain polymorphism.The gene encoding Meta-analysis of observational studies poses particular challenges due to the inherent biases and differences in study designs. In this context, we carried out this meta-analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guideline http://www.wanfangdata.com.cn) and China National Knowledge Infrastructure  databases covering the period from the earliest possible year to March 15, 2013. The following subject terms were used in the search: “advanced glycation end products”, “RAGE”, “AGER”, “coronary heart disease”, “coronary syndrome” or “isch[a]emic heart disease” or “vascular disease” or “myocardial infarction” or “atherosclerosis” or “arteriosclerosis” or “coronary stenosis” or “coronary artery disease” or “coronary disease” or “CAD” or “CHD” or “ACS”, combined with “gene” or “allele” or “genotype” or “polymorphism” or “variant” or “mutation”. The research was also supplemented by reviews of reference lists, hand-searching of relevant journals, and correspondence with authors. Search results were limited to studies on a case-control design and articles published in English or Chinese language.A literature search was conducted of PubMed, EMBASE, Wanfang (Two investigators (F.P. and W.N.) independently obtained the full texts of articles deemed as potentially eligible according to the titles and abstracts. If necessary, we emailed the contributing authors to avoid double counting of participants recruited in more than one study by the same group. Where more than one publication of the same study population existed, we abstracted data from the most recent or most complete publication.Our analyses were restricted to studies that fulfilled the following inclusion criteria : (1) clinical endpoint (dependent variable): CAD or myocardial infarction (MI); (2) study design: either retrospective or nested case-control; (3) independent parameters: either genotypes/alleles of at least one examined polymorphism or circulating sRAGE or endogenous secretory RAGE (esRAGE). Studies were excluded (one was sufficient) if they investigated the progression, severity, phenotype modification, response to treatment or survival, as well as if they were meeting abstracts, case reports/series, editorials, review articles, or non-English and non-Chinese articles.AGER gene, published data were synthesized accordingly.Data were extracted from qualified articles independently by two investigators (F.P. and W.N.) according to a standardized Excel template . Quality assessment was performed in duplicate with κ agreement rate of 0.98. Discrepancies were adjudicated by discussion and a consensus was reached. When three or more studies investigated the same polymorphism in Collected data included the first author, publication year, ethnicity, CAD subtype (CAD or MI), study design, case-control status, genotypes/alleles of examined polymorphisms, circulating sRAGE and/or esRAGE levels, and the demographic records (if available), such as age, gender, matched information, percentage of diabetes and renal disease, body mass index, smoking, systolic and diastolic blood pressure.AGER genetic polymorphisms on CAD. The random-effects model using the DerSimonian & Laird method was employed to calculate weighted odds ratio (OR) and the corresponding 95% confidence interval (95% CI). Comparisons of circulating sRAGE and esRAGE levels between patients and controls were expressed as weighted mean difference (WMD) with 95% CI.To maximize power to detect a true association, only allelic and dominant models were adopted to estimate risk effects of 2 test, and was quantified using the I2 statistic (ranging from 0 to 100%), which is defined as the percentage of the observed between-study variability that is due to heterogeneity rather than chance. Cumulative analyses were performed for all polymorphisms according to the ascending date of publication in order to identify the impact of the first-published article on subsequent publications, and the evolution of the pooled estimates over time.Between-study heterogeneity was assessed by χAGER genetic polymorphisms examined or circulating sRAGE and esRAGE levels on CAD.Predefined subgroup analyses were performed a priori according to the CAD endpoints (CAD and MI), descents of study population , study design (retrospective and prospective), matched information on age and/or gender, and total sample sizes . Meta-regression analyses were performed to evaluate the extent to which different study-level variables, including age, gender, body mass index, smoking, systolic and diastolic blood pressure, explained the heterogeneity of pooled risk estimates of I2 and Egger’s statistics, for which a significance level was set at P<0.1 Publication bias was assessed by the Egger’s test and the trim-and-fill method. The latter was to estimate the number and outcomes of theoretically missing studies resulting from publication bias. P<0.05 was considered statistical significance, with the exception of Characteristics of all qualified studies in this meta-analysis are summarized in Out of 39 qualified groups, 21 included East Asian, 14 included Caucasian, 4 included Middle Eastern, and 1 included African. Seven groups were reportedly matched in age and/or gender between patients and controls. There were 32 groups designed retrospectively and 7 groups prospectively. The patients of 10 groups were clinically diagnosed as MI.AGER gene were examined, including T-429C (rs1800625 in the promoter), T-374A (rs1800624 in the promoter), Gly82Ser (rs2070600 in exon 3) and G1704A (rs184003 in intron 7), and their results were extracted and synthesized in this meta-analysis. In detail, there were 10 (patients/controls: 1945/2013), 14 (2796/2209), 14 (2145/4966), and 3 (1075/1173) groups evaluating the association of these four polymorphisms with CAD. With regard to circulating sRAGE and esRAGE, there were respectively 13 (patients/controls: 1578/1275) and 8 (1752/1860) groups.Four polymorphisms of AGER gene four polymorphisms in association with CAD under allelic and dominant models  under both models, in populations of African descent under dominant model, in studies under retrospective design and involving more than 300 subjects . As for Circulating sRAGE levels were significantly lower in CAD patients of Caucasian descent and in prospectively-designed studies than controls . With reAGER and its circulating forms, cumulative risk estimates tended to be stable with accumulating data over time under both models (data not shown).Regarding four examined polymorphisms of To explore the extent to which study-level variables explain heterogeneity among individual estimates, a set of meta-regression analyses were undertaken. Unfortunately, none of the confounders including age, gender, body mass index, smoking, systolic and diastolic blood pressure could explain large part of heterogeneity for all examined polymorphisms and circulating sRAGE and esRAGE levels (data not shown). Because meta-regression analyses involved studies of limited sample sizes, it might be underpowered to detect a small or moderate effect.AGER genetic polymorphisms and circulating sRAGE and esRAGE levels with the risk of developing CAD. The two noteworthy findings of this study were that (1) despite the overall null association, there was a contributory role of common variants in AGER gene to CAD in patients with diabetes mellitus or renal disease; (2) circulating esRAGE might be a powerful negative predictor for the development of CAD. Moreover, our findings demonstrated that the existence of diversity of ethnicity, study design, case-control matched information and sample size across studies might result to the presence of heterogeneity.On the basis of 27 studies involving 7585 CAD patients and 9240 controls, we evaluated the association of AGER three genetic polymorphisms  and the risk of CAD, and they failed to observe any suggestive association AGER genetic variants on CAD were strikingly potentiated in patients with diabetes mellitus or renal disease. As indicated by clinical investigations, over-expression of AGER gene can enhance inflammatory reaction and matrix metalloprotease expression in plaque macrophages of diabetic patients AGER expression was found to be closely associated with the worsening of chronic kidney disease AGER.More recently, Wang and colleagues have synthesized data from 17 studies on AGER mRNA, constituting approximately 20% of sRAGE levels in humans To shed some light on this hypothesis, we further evaluated the relation between circulating RAGE forms, which can serve as RAGE blockers and might be applicable to human diseases, and CAD, and it is worth noting that circulating esRAGE might be a powerful negative predictor of CAD, even with the presence of diabetes mellitus or renal disease. Specifically, esRAGE is an alternative splicing product of Heterogeneity in a meta-analysis is mostly produced by differences in study-level characteristics. Our subgroup analyses indicated that ethnicity, study design, matched information and sample size might be potential sources of heterogeneity. For example, risk estimate of 82Ser allele on CAD was 1.29 in East Asians, but was 0.9 in Caucasians. A possible explanation may be due to divergent genetic backgrounds or linkage patterns, and usually a variant is in close linkage with another nearby causal variant in one ethnic group but not in another AGER gene, and did not cover other CAD-susceptibility genes, such as angiotensin II receptor, type 1 Meta-analysis is a powerful tool to summarize results of individual studies; however, it is important to recognize certain limitations. First, most qualified studies were retrospective in design, precluding further comments on causality. Second, albeit low probability of publication bias in this meta-analysis, potential selection bias cannot be ruled out, because we only retrieved articles published in English or Chinese. Third, heterogeneity persisted in some subgroups, limiting the interpretation of our pooled estimates. Fourth, as most studies in this meta-analysis enrolled subjects aged more than 50 years, large studies in a younger population of CAD patients are of special interest, because genetic factors may exert great contribution to those in whom CAD develops at a younger age and in the absence of strong environmental risk factors AGER genetic polymorphisms with CAD was potentiated in patients with diabetes mellitus or renal disease. From a practical standpoint, circulating esRAGE might be a powerful negative predictor for the development of CAD. Nevertheless, we hope that this study will establish background data for further investigations into the mechanisms of AGER gene and relevant pathway genes in the development of CAD.Taken together, our findings collectively demonstrated that association of Table S1Checklist of items to include when reporting a systematic review or meta-analysis.(DOC)Click here for additional data file.


Methods of diagrammatic modelling have been greatly developed in the past two decades. Outside the context of infectious diseases, systematic use of diagrams in epidemiology has been mainly confined to the analysis of a single link: that between a disease outcome and its proximal determinant(s). Transmitted causes ("causes of causes") tend not to be systematically analysed.The infectious disease epidemiology modelling tradition models the human population in its environment, typically with the exposure-health relationship and the determinants of exposure being considered at individual and group/ecological levels, respectively. Some properties of the resulting systems are quite general, and are seen in unrelated contexts such as biochemical pathways. Confining analysis to a single link misses the opportunity to discover such properties.The structure of a causal diagram is derived from knowledge about how the world works, as well as from statistical evidence. A single diagram can be used to characterise a whole research area, not just a single analysis - although this depends on the degree of consistency of the causal relationships between different populations - and can therefore be used to integrate multiple datasets.Additional advantages of system-wide models include: the use of instrumental variables - now emerging as an important technique in epidemiology in the context of mendelian randomisation, but under-used in the exploitation of "natural experiments"; the explicit use of change models, which have advantages with respect to inferring causation; and in the detection and elucidation of feedback. In,4. In3,4mechanism that by its operation makes a difference 7]8][7][8]4][[8][7][8][7][8]4]10]11][[11][10][[[11][10]DAGs are composed of variables connected by arrows , but it is not always clear when these are intended to denote a causal relationship or only a probabilistic one. Figure Furthermore, the DAG tradition has its limitations: once one goes beyond the technical issues of inferring the causal status of a particular observed association, other considerations come into play. These require the use of other diagram-associated methods, including the modelling of infectious disease outbreaks with differential equations, fitting statistical models to causal networks, and analysing systems characterised by feedback. The wider properties of such systems are scientifically and practically important, yet are insufficiently appreciated in most of epidemiology.In this paper, we discuss different types of causal system that are relevant to epidemiology: models of infectious disease transmission, in which the human population is located within a broader system with which it interacts; models that integrate the emission and dispersion of pollutants with their impacts on health; and the relationship of social factors to specific risk factors and to selection effects. We describe how diagrams can be employed to improve the analysis of such systems, and in the course of doing so we note that generic properties of the systems can be observed that are independent of the specific content, even though the diagrams themselves have been constructed solely from empirical evidence - no structure has been imposed on them.We draw on a number of traditions that have analysed systems and/or that have used causal diagrams. The most important of these are:• path diagram analysis, which was devised by the geneticist Sewall Wright but which has mainly been employed in quantitative social science analysis, and• the similar but more general method of structural equation modelling, which also systematically analyses measurement error  - includ• econometrics, in which the structure of a system is represented by an equation for each link, albeit without the systematic use of causal diagrams ;• diagrams of metabolic pathways in biochemistry;• the tradition of infectious disease epidemiology modelling , which i• a group of traditions in systems modelling, including cybernetics, dynamical systems modelling, and system dynamics , as wellAnopheles mosquito, and subsequently received the second Nobel prize for medicine. He then defined a mathematical model describing the time dependent dynamics of infection and recovery in human and mosquito populations. The major terms in the differential equations describing this human-mosquito-parasite ecology were : the number of newly infected humans arising due to bites from infected mosquitoes, the number of new mosquito infections due to biting infected humans, and the rate of recovery of both humans and mosquitoes from infection . Her. Her32].The models considered so far have been concerned with only one causal pathway. However, epidemiology of non-infectious diseases usually deals with a situation of multiple causation, in which all (or most) links are analysed as stochastic - there are no necessary or sufficient causes, and Koch's postulates do not apply. Under such conditions, diagrammatic models are no longer confined to a single chain.It is simple to draw a diagram that contains branches, but this introduces new issues that go beyond the scope of the present paper. In principle, causal diagrams and DAGs can readily cope with multiple causation, but further methodological work is needed on effect modification -36.In social epidemiology, a classic question is, how much of the observed social gradient is mediated by known risk factors? It is possible to investigate this question on the simple assumption that no effect modification or other complicating factor is present, in which case a diagram is probably not necessary. However, such an assumption may not be justified. For example, an econometric analysis of the Whitehall II Study has shown that if allowance is made for selection effects, the findings change. Whilst childhood socioeconomic circumstances are still found to impact on adult health, it emerges that the association of current civil service grade with health status reflects the tendency for healthier people to be promoted. And employment grade is also predicted by childhood socioeconomic position, which thus influences adult health both directly and via job success - for example, promotion is more likely for taller people, and height is an indicator of childhood wellbeing .Moreover, a diagram with multiple and branching chains can readily be expanded to encompass a larger system, so enabling integrated analysis of the inter-related factors. In this case the upstream causes can include the wider determinants of ill-health as well as more concrete mediating factors - the "web of causation" for a particular health issue, a concept that has a long history ,39  has/have been constructed, it/they can be used as the framework for statistical analysis of the component links. If in doubt, a postulated link should be included, as it can always be deleted in the light of evidence suggesting its magnitude is zero, whereas discovery of a link that was omitted in error is more difficult - although this can be achieved by algorithms incorporated in software e.g. in the context of DAGs used in genetics. The same applies to variables: they should be included, with all the pathways thought to be possibly relevant, unless and until analysis shows them to be unimportant.  Thus the most conservative diagram contains all possible variables and pathways. The statistical analysis then results in deletion of some links, and quantification of those that remain.In the deletion of links, it is clearly inappropriate to use a simple criterion such as striking out those that do not reach statistical significance. This is because a relationship could fail to reach significance merely due to small sample size. A better method is to use model comparison/selection methods such as those based on likelihood ratio (e.g. Akaike Information Criterion (AIC), or its Bayesian alternative the Deviance Information Criterion (DIC)). However, this process is fallible, especially in the presence of measurement error. An alternative is the use of structural equation modelling, in which latent variables can be introduced to deal with measurement error. The addition of a hierarchical layer modelling the relationship between observations and "true" values of a parameter could be considered, thus defining a hidden Markov Model .In some situations, a causal diagram can become large and complicated, and the quantification of its constituent links may rely on more than one dataset. The diagram then needs to be broken up into smaller components, with the risk of potentially introducing confounding or other distortions. However, this can be overcome if it is possible to use the conditional independence properties specified by the structure of the diagram: if two variables are connected to each other only via a third variable in one of the three ways depicted in Figure One of the distinctive features of a diagrammatic approach is that a causal pathway can be modelled using any parametric form, therefore separating the two key questions "does a link exist?", and "if so, what is its functional form?". This has an advantage over the specification of the system in terms of equations, where the elision of these two questions may be harder to avoid. For example, it is rather straightforward to draw a diagram such as that shown in Figures On the other hand, it is necessary to be cautious - diagrams may make the situation look simpler than it really is. An example of this is transmissibility: it may appear that if X → Y and Y → Z, then it is necessarily true that X → Y → Z. Logically it seems undeniable, but in real life this is not always the case. For example, in toxicology it is typically found that the dose-response relationship has a threshold: below a certain dose of the substance it has no impact on the organism. If this is represented by Y → Z, and the pathway X → Y does not result in the accumulation of Y to the threshold level, then X → Y → Z will not be true. This has fundamental implications even for basic data handling. For example, in studying the possible effects of disinfection by-products on the outcome of pregnancy, it was found that swimming led to infrequent but very high exposure levels . If the One of the most intractable problems with epidemiology, other than in the  situations where randomisation can be used, is that it is difficult to reliably infer causation from observational studies, because the upstream causal pathways are complicated and may introduce confounding or selection. One approach is to try and map out these pathways and analyse them in their own right. An alternative, which also involves analysing a system that is conveniently portrayed by means of a diagram, is to use the instrumental variable approach . The basThe principle is that one or more additional variables - "instruments"- are introduced, associated with the putative causal variables, but not directly with the outcome variable or any potential confounders. Further assumptions are that effect modification and alternative pathways are absent. All these assumptions need to be checked, and a convincing case made that they are satisfied; it is impossible for this to be conclusively established, a similar situation to the familiar case of unmeasured confounding. This approach is the equivalent in observational studies of analysis by intention to treat in randomised controlled trials.A frequently-used method of statistical analysis has two stages: first, the instrument is used in a regression to obtain an estimated value of the putative causal variable, and then this estimated value is plugged into a second regression equation that contains the variables of substantive interest. The estimated value is an unconfounded measure if the above assumptions are met.ALDH2 that a person inherits is strongly associated  with the extent to which they enjoy heavy alcohol consumption, as already stated above, but is unlikely to be associated with the psychosocial factors that may be causes or effects (or both) of the level of alcohol consumption. The polymorphism for this gene can therefore be used as an instrument: it is a cause of the level of drinking that can be assumed to be independent of the various psychosocial and economic factors that would likely introduce confounding . The. The51].founding .predictor of disease even when it is not on the causal pathway. But for intervention it is essential to know whether or not it is on the causal pathway; if it is merely epiphenomenal, then intervening to reduce it will have no effect. Figure This approach has been applied to the use of biomarkers: a biochemical measurement such as plasma C-reactive protein (CRP) can be a useful ALDH2 polymorphism - if the assumption is sustainable that there is no effect modification with other variables in the system.Such approaches can also be used in observational studies that do not involve genetics, as has long been routine in econometrics. A nice example is a study of the effect of family size on the mother's work status: to distinguish a direct causal effect from confounding (e.g. her preference for career as against childbearing) and from reverse causation , the authors used the sex of the first two children as a natural experiment . If theyIt is usual to construct diagrams in terms of the levels of the relevant variables, but an alternative is to instead use their changes. The mathematics of a change (or "first difference") model are different from one in terms of levels, a distinction that is very familiar in econometrics. One advantage is, any elements that remain invariant do not feature in a change model, so it can be a great deal simpler and thus more tractable. This invariance condition can be violated, for example in the presence of effect modification, or when the variable itself has a time-varying effect, such as the differing effect of maternal education on a child's IQ at different ages.A second benefit is that interpretation is clearer: for example, it is relatively straightforward to think about the health impacts of a factory closure, whereas a discussion of the effects of (un)employment on health is more complicated, e.g. due to (self-)selection effects. Evidence derived from a change perspective may also carry more weight causally: for example, a controlled before-after study of a coal ban in Dublin showed the change in pollutant levels and in subsequent mortality there, but not in the rest of Ireland that was unaffected by the ban . This isAn additional benefit of using a change model is that it fits naturally with a focus on intervention Figure . Here thFeedback may sometimes be important. A simple example is in the case of an "accident black spot": if the road design is improved so as to reduce the risk, drivers may respond by increasing their speed, thereby undoing some of the benefit - "risk compensation"  - an exaReinforcing (positive) feedback may also occur, for example, people who are physically inactive may tend to become obese and have other physical changes that further discourage them from exercising, and conversely, more active people have physiological changes that encourage them to take more exercise . Reinforcing feedback also frequently occurs in models of population growth, and therefore in infectious disease modelling which is derived from demography and ecology. For example, this occurs when parasites sexually reproduce : as the parasite population size increases, assuming that the sex ratio remains fixed, so does the likelihood that males and females will discover and mate with one another and therefore population growth occurs faster .More generally, as humans tend to respond systematically to their situation, if the response (e.g. policy or other intervention) is included in the model, then feedback may have to be taken into account. This is true also of conditions  that involve a large behavioural element, such as obesity, mental health and homicide .Vensim [In general, analysing systems with feedback requires a different approach, with diagrams that contain cycles. In infectious disease epidemiology, this is explicit because feedback loops are the rule and acyclic diagrams the exception. Infectious disease epidemiology models are, in general, system dynamical in this sense, and off-the-shelf software such as Vensim  is oftenVensim . An impoVensim ,64, a siFrom the viewpoint of intervention, it is important to recognise when feedback is occurring, particularly because the usual methods of thinking, epidemiological and other, tend not to see it. Thus, compensating feedback frequently occurs, like the risk compensation example, and frustrates policy interventions. And reinforcing feedback - often dismissed pessimistically as a "vicious circle" - can be an opportunity: in the context of absolute poverty, health influences the economic wellbeing of a household, and the latter in turn is a "fundamental determinant" of health . The impExplicitly causal methods of diagramming and modelling have been greatly developed in the past two decades. However, use of such methods in epidemiology has been mainly confined to the analysis of a single link: that between a disease outcome and its proximal determinant(s). Apart from in the context of infectious diseases, they have been under-exploited in their potential to model the larger system in which health is generated or undermined.This approach would accord with wider developments in biology. The Human Genome Project has revealed that the number of protein-coding genes is far fewer than was previously thought, and that they are influenced by upstream genes in that large proportion of DNA that was previously referred to as "junk" . "CausesDiagrams and models are constructed to fit each situation, from a combination of substantive knowledge and statistical evidence - but can then take on properties that result from their abstract structures. By constructing diagrams of a larger system, inter-relationships of different factors can readily be visualised, and then analysed statistically. As well as its scientific function, this has practical advantages in terms of designing interventions.Such methods are applicable to all branches of epidemiology, including infectious diseases epidemiology, chronic disease epidemiology, environmental and occupational epidemiology, and social epidemiology - and especially to their inter-relationship, e.g. simultaneous consideration of social and environmental influences.The authors declare that they have no competing interests.All the authors contributed substantially to the writing of the paper, the drafting and the process of critical revision. The main drafting was carried out by MJ, with important contributions by MG, MC-H and PV according to their particular range of expertise. All authors have read and approved the final manuscript.