MAD1L1 Arg558His and MAD2L1 Leu84Met’s conferring susceptibility to CRC. Cytokinesis-block micronucleus cytome assays were applied to assess the effect of two functional variants on chromosomal instability (CIN). Significant associations with CRC risk were observed for MAD1L1 Arg558His  and MAD2L1 Leu84Met in a dominant model . Moreover, significant multiplicative gene-smoking interactions were found in MAD1L1 Arg558His (P = 0.019) and MAD2L184 Leu/Met (P = 0.016) to enhance CRC risk. Additionally, the frequencies of lymphocytic micro-nucleated binucleated cells for MAD1L1 Arg558His polymorphism were significantly different in the exposed group (P = 0.013), but not in the control group. The study emphasized that MAD1L1 Arg558His and MAD2L1 Leu84Met can significantly interact with smoking to enhance CRC risk, and the genetic effects of MAD1L1Arg558His on CIN need to be further clarified in follow-up studies.The spindle assembly checkpoint (SAC) has been established as an important mechanism of driving aneuploidy, which occurs at a high frequency in the colorectal tumorigenesis. Two important components of SAC are MAD1L1 and MAD2L1, which function together in an interactive manner to initiate the checkpoint signal. We hypothesize that genetic variants in the binding domains of MAD1L1 and MAD2L1 may modulate protein structures and eventually contribute to CRC susceptibility. A case-control study including 710 CRC cases and 735 controls was performed to examine  Colorectal cancer (CRC) remains the third most commonly diagnosed cancer and the fourth leading cause of cancer mortality worldwide, accounting for 8.3% of the total cancer cases and 6.3% of cancer deathsGenome instability is the prominent hallmark of human cancers and of chromosomal aneuploidy, which is the most common type of genomic instability, occurring at a high frequency in the colorectal tumorigenesis24Two interactive components of the SAC are the human MAD1L1 mitotic arrest deficient-like 1(MAD1L1) and MAD2L1 mitotic arrest deficient-like 1(MAD2L1), which function together in a hetero-tetrameric complex to initiate the checkpoint signal691113MAD1L1 Arg558His (rs1801368) is a missense variant at codon 558 that is located in the region that encodes the second leucine zipper domain of MAD1L1, and the His allele of MAD1L1 Arg558His was identified to reduce the binding activity of MAD2L1 to MAD1L1, with the result of a decreased proficiency in enforcing mitotic arrest. Moreover, MAD1L1 Arg558His has been previously reported to be associated with lung cancer risk1584 to Met84 substitution in the MAD2L1 molecule has also been found to influence the interaction between MAD1L1 and MAD2L1MAD1L1 Arg558His and MAD2L1, Leu84Met may play an important role in transducing the checkpoint signal by potentially influencing the protein activity. An alternative hypothesis has been proposed that MAD1L1 Arg558His and MAD2L1 Leu84Met may be associated with the risk of CRC.Therefore, the binding domains of MAD1L1 and MAD2L1 are required for transducing the checkpoint signal, and any change in the gene structure may disturb the process of checkpoint signal transduction. Genetic variants in the binding domains of MAD1L1 and MAD2L1 may modulate protein structures and eventually contribute to CRC susceptibility. MAD1L1 Arg558His and MAD2L1 Leu84Met variants may result in a mitotic check-point defect by severely disturbing the domains of MAD1L1 and MAD2L1, directly leading to chromosomal instability (CIN). It has been clear that CIN is the important phenotype of the carcinogenesis and that it plays a causative role in tumor initiation and progression in CRC biology. Consequently, not only were epidemiology studies conducted to explore the association between the two variants and the risk of CRC, but also cytokinesis-block micronucleus cytome assays (CBMN) have been applied to examine the effect of two functional genetic variants on chromosomal instability (CIN). Here a case-control study consisting of 710 CRC cases and 735 controls was performed to examine MAD1L1 Arg558His or MAD2L1 Leu84Met conferring susceptibility to CRC in a Chinese Han population. Moreover, the frequencies of lymphocytic micronucleated binucleated cells (MNBNCs) in peripheral lymphocytes was used to assess the effect of the two candidate SNPs on the formation of aneuploidy.Biological evidence has indicated that the P = 0.520) and age group (P = 0.228). Smoking was significantly associated with an increased risk of CRC (P < 0.001), with 64.1% of them being smokers and 54.6%, controls. Furthermore, a significant difference in the smoking level was observed between the case and the control groups (P < 0.001). Among the smokers, the heavy smokers—who smoked >24 packs per year—were overrepresented compared with the controls (64.0% vs 49.1%). Compared with nonsmokers, the OR for smokers was 1.95 (95% CI = 1.49–2.54).The baseline characteristics of 710 CRC patients and 735 controls are shown in P = 0.872 for MAD1L1 Arg558His, P = 0.311 for MAD2L1 84Leu/Met). Minor allele frequencies (MAF) of MAD1L1 Arg558His and MAD2L1 84Leu/Met were 0.461 and 0.059 in controls and 0.524 and 0.087 in cases, respectively. In the logistic regression analysis, the two candidate SNPs were independently associated with CRC risk after adjusting for age, sex, and smoking status. Compared with individuals carrying the MAD1L1Arg/Arg genotype, individuals with Arg/His genotype presented a significantly increased risk of CRC on the borderline level , but a significant association with CRC risk was observed for individuals with the His/His genotype, with OR of 1.70 (95% CI = 1.26–2.28). For MAD2L1 84Leu/Met SNP, heterozygous or homozygous variant genotypes significantly increased the CRC risk compared with the homozygous wild type in dominant genetic models, with OR equal to 1.48 (95% CI: 1.09–2.01).The genotype information of the two candidate SNPs are presented in MAD1L1 Arg558His and MAD2L1 84Leu/Met on the risk of CRC. Compared to individuals who carried the combination of MAD1L1 Arg/Arg and MAD2L1 Leu/Leu genotypes, we observed that individuals with MAD1L1 His/His and MAD2L1 Leu/Met or Met/Met genotypes had an increased risk of OR for CRC risk, up to 2.92 (95% CI: 1.63–5.24). However, no significant multiplicative or additive gene-gene interactions were observed between MAD1L1 Arg558His and MAD2L1 84Leu/Met on the risk of CRC.In the present study, the stratified analysis and multiplicative and additive interaction analyses were performed to explore the relationship between the two SNPs and smoking . When stP = 0.580); however, a significant result emerged in the exposed group. MNBNC frequencies for individuals with MAD1L1 His/His, Arg/His genotypes and Arg/His genotypes in the exposed group were 3.83 ± 2.58(‰), 4.32 ± 2.68(‰) and 6.67 ± 2.99(‰), respectively, with the P value equal to 0.013. Furthermore, negative results were also found for MAD2L1 Leu84Met in both the exposed and the control groups, with no significantly different distribution of MNBNC frequencies for the three genotypes.Both MNBNC frequency and genotypes for the two candidate SNPs were examined in the exposed group and the healthy group. MAD1L1 Arg558His or MAD2L1 Leu84Met with the risk of CRC in a Chinese population. The study demonstrated that both MAD1L1 Arg558His and MAD2L1 Leu84Met significantly increased the risk of CRC. Moreover, significant multiplicative gene-smoking interactions were found for MAD1L1 Arg558His and MAD2L1 84Leu/Met to enhance the risk of CRC. In addition, a significant statistical difference was observed for the effect of MAD1L1L1 Arg558His on the formation of aneuploidy in the exposed group, but not in the healthy control group.In this study, we conducted a case-control study to explore the association of MAD1L1 Arg558His cause a deficient metaphase arrest in normal cellsMAD2L1 84Leu/Met may reduce the spindle checkpoint function by physically affecting the interaction of MAD2L1 with MAD1L1 as illustrated via the Co-IP assaysMAD1L1 558His and MAD2L1L1 84Met are associated with the increased risk of CRC. However, an association of SAC components on CRC risk have not appeared from much larger GWAS studies. It can be given the explanations that the GWAS approach relies on randomly selected SNPs across the genome as markers, most of the associated SNPs identified thus far are unlikely to be the actual causal variants. Besides, in order to prevent false positives associated, GWAS analysis using very stringent P values, it is possible to miss some of the P value which is not reached GWAS request is indeed associated SNP20MAD1L1 Arg558His and MAD2L1 Leu84Met with smoking to increase the risk of CRC. One previous finding from De Voer et al. indicated that germ-line mutations in the SAC genes BUB1 and BUB3 are associated with an increased risk of CRCet al.MAD1L1 and MAD2L1, were found not to be significantly associated with the risk of familial breast cancer risk. Further epidemiology studies can also be performed to confirm the results and to estimate the effect of the two missense variants on the risk of other cancers.The spindle checkpoint is an intricate multi-protein network that regulates the attachment of sister chromatids to the mitotic spindle in the metaphase to anaphase transition. The cell will proceed to aneuploidy when improper attachments of chromosomes to the spindle microtubules happen. Previous evidence has shown that the chromosomal instability that is induced by the deregulation of the mitotic checkpoint gene can been widely observed in colorectal cancers1718MAD1L1 Arg558His and MAD2L1 Leu84Met, in the present study, we observed no significant interaction between them with regard to the risk of CRC, and a similar result was also found on the risk of lung cancer in a previous studyMAD2L1 and MAD1L1 are two interactive proteins that are involved in stimulating and executing spindle checkpoint processes. Although biological analyses have shown an attenuated interaction between MAD1L1 Arg558His and MAD2L1 84Leu/Met to enhance the risk of CRC. Smoking has been firmly established as an important causal factor for the development of CRC by a mass of evidence23MAD1L1 Arg558His, MAD2L1 84Leu/Met and smoking. However, it has been reported that the important specified carcinogens of cigarette smoke, nicotine and polycyclic aromatic hydrocarbons may induce the process of genomic instability by various molecular mechanisms252627MAD1L1 Arg558His, MAD2L1 84Leu/Met and smoking.In the present study, the most significant finding was that smoking may interact with MAD1L1 Arg558His or MAD2L1 84Leu/Met on the formation of aneuploidy. Remarkably, MNBNC frequencies for individuals with MAD1L1 His/His, Arg/His genotypes and Arg/His genotypes were significantly different in the exposed group, but not in the healthy control group. MNBNC frequency is an important indicator to assess the level of CIN. The biological evidence has indicated that the MAD1L1 Arg558His may indirectly lead to CIN by severely disturbing the function of the mitotic check point. As we know, the genetic effect of the variants on the important phenotype is modest, and it could be detected by a study with a sufficiently large sample size. However, when exposed to some pollutants like e-waste, the genetic effect of variants can emerge by the induction of a toxicant. Therefore, it can be concluded that MAD1L1 Arg558His may contribute to CIN potentially through environmental exposure. It can also be speculated that the negative result in the healthy control group may be due to the relatively low statistical power of detecting the modest effect of the potential functional SNPs. Furthermore, the significant effect of MAD1L1 Arg558His on the formation of aneuploidy was also observed in a previous studyMAD1L1 Arg558His on chromosomal instability in healthy subjects with enough sample size. However, the negative effect of MAD2L1 84Leu/Met on the formation of aneuploidy as found in both the exposed and in the healthy control groups. Therefore, independent replication studies with a larger sample size are warranted to verify the negative result.Lastly, the study explored the effect of MAD1L1 Arg558His and MAD2L1 Leu84Met, and the effect of MAD1L1 Arg558His or MAD2L1 Leu84Met on the formation of aneuploidy. Besides, because of the epidemiological background, we have not addressed the biological functional analyses to dissect the molecular mechanism underlying the significant statistical interaction between MAD1L1 Arg558His, MAD2L1 84Leu/Met and smoking. Despite these limitations, our study emphasized that MAD1L1 Arg558His and MAD2L1 Leu84Met variations confer susceptibility to CRC in a Han Chinese population. Also, our findings highlight the point that two variations can significantly interact with smoking to enhance the risk of CRC. The information of the smoking level was detailed and complete, so the OR for MAD1L1 Arg558His and MAD2L1 Leu84Met that are stratified by different smoking levels were able to be estimated in the present study. In addition, the study showed that MAD1L1 Arg558His may contribute to CIN potentially through the induction of environmental exposure. Nevertheless, follow-up studies are needed to uncover the biological mechanism behind the significant statistical interaction between MAD1L1 Arg558His, MAD2L1 84Leu/Met and smoking, and further clarify the genetic effect of MAD1L1 Arg558His on chromosomal instability.The present study has addressed some limitations, and it has some advantages. First, the sample size of the study was not very large, and the statistical power is relatively low for detecting the gene-to-gene interaction between The present study included 710 CRC patients with newly diagnosed CRC and 735 cancer-free controls. Patients were consecutively recruited between January 1, 2010 and November 31, 2013 at Tongji Hospital of Huazhong University of Science and Technology (HUST), Wuhan, central China. Cancer-free individuals living in Wuhan city and its surrounding regions were randomly selected from a healthy screening conducted at the same hospital during the same time. All subjects were unrelated Han Chinese. The inclusion criteria for patients included histopathologically confirmed CRC, without previous chemotherapy or radiotherapy, and no restriction in regards to sex, age, or disease stage. The selection criteria for controls included cancer-free individuals and frequency matched to cases for sex and age within five years. At recruitment, written informed consent was obtained from each subject, and blood samples and demographic characteristics, including sex, age, and smoking habits, were collected by the interviewers. The detailed definitions of smoking status were described previouslyThe participants who were included in the CBMN to estimate the genetic effect of two functional genetic variants on chromosomal instability were selected from a typical e-waste recycling site in southeast China as the exposure group and a nearby village (50 km away from the exposure site) without known sources of industrial pollution as the control groupMAD1L1 Arg558His or MAD2L1 Leu84Met sites were described previouslyMAD1L1) or 62 °C (for MAD2L1) for 30 seconds, 72 °C for 45 seconds, and a final elongation step of 72 °C for 10 minutes. Restriction enzymes BstUI or AlwNI  were used to distinguish the MAD1L1 Arg558His or MAD2L1 Leu84Met genotypes, respectively. Quality control was monitored by including a 5% duplicate, with a 100% concurrence rate of the duplicate sets. The average call rate for the candidate SNPs genotyped was >99%.Genomic DNA was extracted from 2 ml of peripheral blood samples using the approved guideline of the Relax Gene Blood DNA System DP319-02 . Candidate SNPs were genotyped using PCR-based restriction fragment length polymorphism (RFLP) methods which were carried out in accordance with the approved guidelines, without knowledge of the case or control status of the subjects. The PCR primer pairs used for amplifying DNA containing the Genomic DNA was extracted from the 2 ml of peripheral blood samples using the Relax Gene Blood DNA System DP319-02 . Candidate SNPs were genotyped using the TaqMan real-time polymerase chain reaction (PCR) Assay  without knowledge of the case or control status of the subjects. The PCR program included heating to 95 °C for 10 minutes followed by 50 cycles of 92 °C for 15 seconds and 60 °C for 90 seconds. The ABI Prism 7900HT Sequence Detection System was applied to read the reacted plates and to analyze the endpoint fluorescence. Quality control was monitored by including a 5% duplicate and negative control, with a 100% concurrence rate of the duplicate sets. The average call rate for the candidate SNPs genotyped was >99%. All the experimental protocols were approved by TIANGEN BIOTECH (BEIJING) CO., LTD and New England Biolabs, MA, USA.MAD1L1 Arg558His and MAD2L1 Leu84Met, and the corresponding MNBNC frequency. The genotype method was described in the previous paragraph. The frequency of MNBNCs was detected by the method that was carried out in accordance with the approved guidelines and was described in detail in the previous study70 independent exposed subjects and 70 control subjects were selected to determine the genotypes of The Hardy-Weinberg equilibrium (HWE) for genotypes was assessed by a goodness-of-fit X2 test in the control groups. In the baseline analysis, the X2 test was applied to examine the differences between cases and controls in distribution of sex, age, smoking status and smoking level. The one-way ANOVA was used to determine the differences of MNBNC frequency for individuals with different genotypes. Co-dominant, dominant, and additive genetic models were assumed in the association analysis. Because minor allele frequencies (MAF) of MAD1L1 Arg558His and MAD2L1 84Leu/Met were 0.461 and 0.059 in controls, we calculated the power for the sample size of 710 patients and 735 controls as follows: for SNP with MAF of 0.059, the power for our sample size to detect an OR of 1.50 was 0.51; for SNP with MAF of 0.461, the power to detect an OR of 1.50 is 0.97. Interaction between SNPs was subsequently systematically investigated by a pair-wise analysis under multiplicative and additive interaction models. The P-values for multiplicative interaction were calculated using a multiplicative interaction term included in the multivariate logistic regression model in SPSS software. The P values for the additive interaction were calculated by a bootstrapping test of goodness-of-fit of the null hypothesis for no departure from an additive model vs. an alternative hypothesis for a departure from an additive model using Stata 10.0 .How to cite this article: Zhong, R. et al. MAD1L1 Arg558His and MAD2L1 Leu84Met interaction with smoking increase the risk of colorectal cancer. Sci. Rep. 5, 12202; doi: 10.1038/srep12202 (2015).


CCND1 gene affect the cell cycle and are frequently observed in a variety of cancers. While the most frequent mutations that occur in thyroid tumor tissue have been characterized, the genetic factors that predispose individuals to differentiated thyroid cancer (DTC) remain to be elucidated. The present study examined whether the CCND1 c.723G>A  and c.669C>T  variants have an impact on DTC susceptibility. A cohort consisting of 652 patients diagnosed with DTC were analyzed and comapred with a reference group of 799 subjects from the general population. Pyrosequencing was used as the genotyping technique. In order to determine the statistical significance of differences observed in the genotypic and allelic frequencies between the compared groups, GraphPad Prism 4 was used. At the rs9344 locus in the DTC patients, a higher frequency of allele A  and the AA homozygous genotype  was observed compared with the control population group. The differences were stronger for papillary carcinomas , but were not significant in follicular tumors. No statistically significant differences were noted in the frequency of genotypes or alleles at the rs3862792 locus in the examined groups. The present findings indicate that the c.723A variant of the CCDN1 gene may be a susceptibility low penetrance allele in the development of papillary thyroid cancer in the population studied, however it does not impact on multifocality, metastatic ability or age at diagnosis. A cumulative effect of the analyzed CCND1 gene variants was also excluded.Alterations in the  Differentiated thyroid carcinoma (DTC) accounts for ~1.5% of all malignancies in the Polish population, and its incidence is increasing worldwide. This may be due to evolving diagnostic technology and medical surveillance practices, however, the technological advances in medical sciences only partly explain the observed rise in this incidence. The genesis of thyroid cancer is complex and connected with several factors, including exposure to chemical agents or irradiation, iodine deficiency and genetic predisposition , which are activated by the formation of complexes with cyclins. The  S phase .CCND1 gene that altered cell cycle progression have been frequently observed in various tumors and may contribute to tumorigenesis. Germline sequence variants of this gene have been the subject of numerous studies into cancer diseases. The evidence indicates that common single nucleotide polymorphisms (SNPs), which are known to alter the splicing of the cyclin D1 transcript, may modify the action of cyclin D1 in cancer cells and affect the cancer risk and outcome  and c.669C>T , have an impact on the occurrence of FTC and PTC. The present study also aimed to demonstrate an association between the aforementioned SNP allelic variants and DTC, with particular reference to gender, histological subtype, patient age at the time of diagnosis, tumor-node-metastasis staging and the occurrence of metastases.The aim of the present study was to determine if the A group of 647 patients with DTC  from Wielkopolska (Greater Poland) was examined. The gender ratio  was 7.1:1, which varies slightly from previously reported gender ratios . The population group consisted of unrelated individuals from the Wielkopolska region who attended the hospital for paternity testing.All individuals examined in the present study agreed to take part in the genetic testing, in accordance with the requirements of the Ethical Committee of Poznań University of Medical Science (acceptance no. 629/07).DNA was obtained from whole blood leukocytes by guanidine thiocyanate and subsequent phenol-chloroform extraction. Pyrosequencing was used as the genotyping technique.CCND1 gene were as follows: c.723G>A (rs9344) forward, biotinylated 5′-TCCTACTACCGCCTCACACGC and reverse, 5′-GCACTAGGTGTCTCCCCCTGTAA , and sequencing primer, 5′-GGACATCACCCTCACTTA; and c.669C>T (rs3862792) forward, 5′-AATCCGCCCTCCATGGTG and reverse, biotinylated 5′-CGTGTGAGGCGGTAGTAGGAC , and sequencing primer, 5′-AACCTGAGGAGCCCC. The pyrosequencing reactions were performed on a PSQ96 device  using the PyroMark Gold Q96 reagents kit (Qiagen).The sequence of the primers used in the analysis were designed by PyroMark Assay Design Software 2.0 . The primer sequences used in the analysis of the 2 test was used to evaluate the differences in genotypic and allelic frequencies between the groups. Furthermore, statistical analyses that assessed the gender, histological type of the cancer, patient age at diagnosis and metastatic potential were performed.The prevalence of the genotypes in the patients and control individuals was examined by the Hardy-Weinberg equilibrium . The χ2 In order to determine the statistical significance of the differences observed between the compared groups, GraphPad Prism 4  was used to obtain odds ratios (OR), 95% confidence intervals (CI) and P-values. P<0.05 was considered to indicate a statistically significant difference.CCND1 gene was analyzed by Haploview v.3.11  (The linkage disequilibrium between the c.723G>A (rs9344) and c.669C>T (rs3862792) polymorphisms of the MA, USA) .The genotyping results of the c.723G>A (rs9344) locus were obtained by pyrosequencing  for all The difference in the frequency of the alleles and genotypes that was noted in the patients with thyroid cancer appeared to be even more important . A signiDue to an almost seven-fold higher incidence of thyroid cancer in females, statistical analysis was performed separately for each gender. By comparing the females with DTC against those from the population group, an association between genotype AA and thyroid cancer was identified, but no relevant associations in the male patients were noted and there were no statistically significant differences between the genders.In the next step, the histological type of thyroid tumor was focused upon. PTC was observed almost nine times more frequently than FTC in patients with thyroid cancer. Statistical calculations revealed that the minor A allele was present significantly more in the PTC patients and that the association observed for homozygous AA was even more apparent. When females with PTC were compared with the whole population, the statistically significant difference remained. No statistically significant differences were observed for the alleles and genotype frequencies between the patients diagnosed with FTC and the population or control group with nodular goiter.In the analysis of c.669C>T (rs3862792), pyrosequencing results were obtained for 642 DTC patients and 628 individuals from the population.No differences in allele and genotype frequencies were observed. Additionally, the rare TT genotype was absent in the whole analyzed group . A compaStatistical tests were also performed to identify possible correlations between the carried genotype and the age at diagnosis. In the designed age groups, any correlation between the variables in results obtained for the rs9344 and rs3862792 locus analysis were noted .Metastases and recurrences were present in 123 DTC patients (19%). In the patient group, the main metastases observed were to the cervical and mediastinal lymph nodes, lungs and bones. No significant association between the occurrence of metastases and the alleles or genotypes of the two polymorphic sequence variants was noted. There was also no significant correlation between the studied SNP variant presence and tumor size . HoweverCCND1 gene. This analysis was performed using Haploview v.3.11 (Broad Institute)  .2=0.27). The strong linkage disequilibrium enabled the haplotypes for 542 DTC patients and 512 individuals from the population to be determined.The distance between the c.669C>T and c.723G>A polymorphisms is only 54 bp. The two polymorphisms demonstrate extremely strong linkage disequilibrium, with a D′ value of 0.84–1.0, although the correlation coefficient between these two loci does not allow for the clear identification of a single allele of one polymorphism based on information from the second SNP allele  in exon 4 is one of the most frequently cited in cancer research. This polymorphism increases the frequency of the alternate splicing of cyclin D1 mRNA; allele c.723A results in a higher level of transcripts encoding a protein with an altered C-terminal domain. This variant may have a longer half-life than the c.723G variant, which may cause the G1/S-checkpoint bypass  is located in exon 3 and is a rare polymorphism. This polymorphism has been investigated in studies on ovarian, uterine, cervical  and brea studies . The fre studies . DiffereThe present results suggested that the c.723A variant may be a risk factor for DTC occurrence in homozygotes. The present population-based, retrospective study found that the c.723A variant is associated with an increased risk of PTC.The present findings would require confirmation through large, population-based, unbiased studies of other ethnic groups.


I2=96.7%; P<0.001]. The association was also significant in Asian population  and European populations . After conducting the subgroup analysis and sensitive analysis, the results showed consistent significant association with the one based on all studies. No publication biases were found. Our analysis indicated that plasma or serum copper level in PE patients was significantly higher than that in healthy pregnancy women.The association between copper level and risk of preeclampsia (PE) has produced inconsistent results. Thus, a meta-analysis was conducted to summarize the evidence from epidemiological studies for copper level and PE risk. Pertinent studies were identified by a search of PubMed and Web of Knowledge up to April 2016. Standardized mean difference (SMD) was performed to combine the results. Random-effect model (REM) was used. Publication bias was estimated using Egger's regression asymmetry test. Twelve articles  involving 442 PE cases and 463 health controls were included in this meta-analysis. Our pooled results suggested that PE patients had a higher copper level compared with healthy pregnancy controls [summary SMD=0.69, 95% CI: 0.54–0.84,  Preeclampsia (PE) is defined as a progressive, multisystemic disorder which characterized by triad of high-blood pressure to the extent of 140/90 mm Hg or more, oedema, as well as proteinuria, developing after 20 weeks of gestation . FurtherCopper is an important trace element which takes parts in structure of many enzymes like lysyl oxidase, cytochrome coxidase, tyrosinase, dopamine-β-hydroxylase, peptidylgly-cine alpha-amidating monooxygenase, monoamine oxidase, ceruloplasmin and copper–zinc superoxide dismutase (SOD) . Many diWe performed a systematic literature search from January 1990 to April 2016 using the databases of PubMed and Web of Science, using the following search terms: ‘copper’ and ‘preeclampsia’ to search the English related articles without restrictions. Moreover, we also reviewed the references of the included studies and review articles to identify additional studies which were not captured by our database searches.The inclusion criteria were as follows: (1) observational studies design; (2) the exposure of interest was copper level; (3) the outcome of interest was PE; (4) the blood sample was venous blood taken from cases and controls after the 20th week of pregnancy; (5) data of the plasma or serum copper levels are available in the results or can be obtained from the corresponding author; (6) data were presented as mean ± standard deviation (S.D.) . Accordingly, we excluded reviews, letters, editorials and RCT studies.All identified studies were carefully reviewed independently by two investigators to identify and determine whether an individual study was eligible for inclusion criteria in this meta-analysis. If the two reviewers could not reach a consensus about the eligibility of an article, it was resolved by disputing with a third investigator.. Because the underlying units of measurement varied from study to study, all units were converted to ug/l.Two investigators independently performed the search, reviewing all identified studies and collecting data. The following data were extracted: the mean ± S.D. on plasma or serum copper levels, first authors’ name, publication year, country where the study was performed, follow-up duration, mean age, criteria for PE diagnosis, sample size, fasting status before blood collection, determination method of plasma or serum copper levels and other information. If given the standard error mean (S.E.M.) of copper level in the study, the S.D. is calculated by the following formula: S.E.M.=S.D./I2 was used to assess heterogeneity   and cross-sectional studies . The pooled SMD were significant in studies conducted among Asian populations  and European populations . We also conducted a subgroup analysis stratified by fasting status of participants, the pooled SMD was 0.77  in the subgroup of fasting. The pooled SMD were 0.68  in the serum-based sample subgroup and 0.76  in the plasma-based sample subgroup.P value of Egger test was 0.268.As sensitivity analyses, we excluded one study at a time to assess the stability of the results. There was no significant change in the pooled SMD or 95% CI on excluding any of the studies. This means no individual study had excessive influence on the pooled effect between risk of plasma or serum copper and PE risk. The Egger test showed no evidence of significant publication bias for the analysis between plasma or serum copper level and PE for all included studies. The In recent years, many studies had been performed to evaluate the relationship between blood copper level and PE based on populations. To our knowledge, our study is the first meta-analysis to discuss the association between plasma or serum copper level and PE. Our meta-analysis contained 12 studies, including 463 healthy pregnancy controls and 442 PE cases, identified that the plasma or serum copper level in PE patients was significantly higher than that in healthy pregnancy women.PE is a multisystem and multifactorial disease that affects both mother and foetus by vascular dysfunction and by intrauterine growth restriction . AlthougBetween-study heterogeneity is common in meta-analysis , and it As a meta-analysis of relevant published studies, our study has several strengths. First, the large numbers of participants allow a much greater possibility of reaching reasonable conclusions and conducting subgroup analysis. Second, the literatures search and data extraction was conducted independently by two investigators; therefore, the random error introduced by inserting data into tables was avoided greatly. Third, we set a strict inclusion criterion to control the quality of including studies. Fourth, after conducting the subgroup analysis and sensitive analysis, the results showed consistent significant association with the one based on all studies, strongly identifying the association stable.P value of Egger test was 0.268.However, our study also has limitations. First, significant heterogeneity existed overall. To clarify the sources of heterogeneity, we conducted the subgroup and sensitivity analyses. However, residual confounding factors across studies remain as a cause for concern in this meta-analysis. Therefore, heterogeneity was still an inevitable problem that may affect the precision of overall results. Second, we found significant associations between copper levels with the risk of PE both in the Asian populations and European populations. However, only two studies came from Europe, so, more studies originating in Europe and other countries are required to investigate the association between copper levels and PE risk. Due to this limitation, the results are applicable to the Asia, but cannot be extended to populations elsewhere. Finally, although we only included the article written in English and did not search grey literature, the Egger test showed no evidence of significant publication bias for the analysis between plasma or serum copper level and PE for all included studies. The In summary, results from this meta-analysis indicate that plasma or serum copper level in PE patients was significantly higher than that in healthy pregnancy women.


MUC1 polymorphism rs4072037 and the risk of gastric cancer has been described in several studies. However, these studies yielded inconsistent results, especially in different pathological type of gastric cancer. Therefore, we performed this meta-analysis to evaluate the relationship between MUC1 gene polymorphism and gastric cancer susceptibility. A comprehensive database search was performed to identify eligible studies. Odds ratios with 95% confidence intervals were calculated to assess the strength of the association between MUC1 rs4072037 and risk of gastric cancer. Subgroup analyses, publication bias, and sensitivity analyses were also conducted. PubMed, EMBASE, Web of Science and CNKI databases were systematically searched to identify relevant studies. A total of 9 studies (12 datasets) were included in the meta-analysis including 10,410 cases and 11,437 controls. Overall, the G allele at rs4072037 of MUC1 gene was associated with a significant decreased gastric cancer risk . The association was significant in both anatomic location and pathological subtype subgroup analyses. However, the association was detected in Asian rather than Caucasian. Our findings demonstrate that the presence of the G allele at rs4072037 of the MUC1 gene may contribute to protection against gastric cancer in Asian. Further large studies of multiethnic groups are needed to validate these findings.The association between The online version of this article (doi:10.1186/2193-1801-3-599) contains supplementary material, which is available to authorized users. Helicobacter pylori is the single most important etiological agent in the pathogenesis of GC  is the fourth most common cancer and the second leading course of cancer-related deaths worldwide, with an estimated 989,600 new cases and 738,000 deaths in 2008  and sialic acid-binding adhesin (SabA) to the gastric mucosa, thus limiting H.pylori colonization   were cohort-based or case–control studies, and (iii) reported data necessary to calculate the odds ratios (ORs) with corresponding 95% confidence intervals (95% CIs). If such data were unavailable, attempts were made to contact the first author and/or corresponding author via e-mail to provide the missing data before the study was excluded from the final analysis. The major exclusion criteria included: (i) reviews, case-only studies, or familial studies, (ii) lack of sufficient data for calculation of ORs with 95% CIs, and (iii) duplication of previous publications or replicated samples.Study eligibility was determined independently by two reviewers (LXY and ZXW). Disagreements were solved by consensus. Studies were considered for inclusion if they meet the following criteria: (i) evaluated Data extraction was carried out independently by two reviewers (LXY and ZXW) using a pre-defined standard form. Disagreements were resolved by discussion with a third author (WZC). From each study, the following information was extracted: first author’s name, year of publication, ethnicity of the patients, source of control groups , age, sex, genotyping method, Hardy-Weinberg equilibrium (HWE) of controls, and frequency of various genotypes in cases and controls, adjusted ORs and 95% CIs. If allele frequencies were not given, they were calculated from the corresponding genotype distributions. If genotype frequencies were not given, they were calculated from allele frequencies only when the study was in accordance with HWE. For one study , homozygote (GG vs. AA), heterozygote (AG vs. AA), dominant (GG/AG vs. AA), and recessive (GG vs. AG/AA). If adjusted ORs and 95% CIs were reported based on A allele vs. G allele, they were converted to G allele vs. A allele.ORs and 95% CIs were used to assess the strength of the associations between I2 tests were performed to assess the statistical heterogeneity, and the Q-statistic tests with P< 0.10 were used to define a significant degree of heterogeneity. A fixed-effect model was used when there was no heterogeneity (P ≥0.10 for Q-test) , quality scores (score >7 or ≤7) adjustment and statistical power. Sensitivity analyses were performed by deletion of a single study each time to reflect the influence of the individual data set on the pooled ORs. Begg’s funnel plots  , quality score (score >7 or ≤7), adjustment and statistical power (≥0.8 or<0.8) were performed only under the allele model, which had the largest sample size among all genotypic models Table . SubgrouSubgroup analyses were also performed according to ethnicity. The results indicated that G allele was associated with decreased gastric cancer risk in Asian  rather than Caucasian . Stratification of the Asian subgroup into Chinese, Japanese and Korean did not alter the results. Notably, the subgroup of Caucasian included fewer than three cohorts, which could not yield reliable results in the meta-analysis .Begg’s funnel plots Figure  were conMUC1 polymorphism has been reported to be associated with gastric cancer, several studies were performed to validate this finding. However, the results were inconsistent especially in different pathological type. Therefore, we performed the first meta-analysis to assess the relationship between MUC1 gene polymorphism rs4072037 and gastric cancer susceptibility.The identification of genetic variants capable of modulating cancer development could be helpful for the early detection and design of targeted treatment and prevention strategies. With high interest in gene susceptibility to carcinogenesis, increasing efforts have been devoted to the study of genetic variants and gastric cancer risk. Since MUC1 rs4072037 and gastric cancer susceptibility was detected under all genotypic models without significant heterogeneity, suggesting decreased gastric cancer risk for individuals carrying the G allele. Although some of the included studies did not have enough statistical power, this did not influence the overall results and confidence because in the subgroup analyses, studies with power larger than or less than 0.8 yielded similar results with statistical significance.The meta-analysis included 10,410 gastric cancer patients and 11,437 controls, which was far larger than the sample size of the discovery study and the GWAS. Association of MUC1 rs4072037 was associated with gastric cancer risk in diffuse-type gastric cancer rather than intestinal-type gastric cancer. However, our meta-analysis detected significant association in both subtypes. The sample size of the intestinal-type gastric cancer subgroup in our meta-analysis was twice larger than that in the Japanese study, indicating that our result was solid.A large-sample-size Japanese study  rather than Caucasian , which is different from the conclusion of Zheng et al. Additional file 1:Characteristics of included studies.(DOC 49 KB)Additional file 2:Forest plots describing subgroup analyses ofMUC1rs4072037 and susceptibility to gastric cancer under the allele model.(DOC 714 KB)Additional file 3:


P < 0.001), and the hepatic lipase (LIPC) rs10468017 variant was associated with a significantly decreased risk of AMD . Individuals carrying the lipoprotein lipase (LPL) rs12678919 polymorphism (A → G) had no significant change in the risk of developing AMD . After adjusting for the complement factor H (CFH) gene, both CETP and LPL conferred a significantly increased AMD risk . Subgroup analysis based on ethnicity revealed a significant association between the CETP variant and AMD in both Americans  and Europeans . This meta-analysis revealed that both CETP rs3764261 and LIPC rs10468017 polymorphisms were significantly associated with AMD risk. After adjustment for the CFH gene, CETP/LPL conferred a significantly increased susceptibility to the disease, indicating potential interactions among genes in the complement system and the lipid metabolism pathway.Three high-density lipoprotein (HDL)-related loci have been reported to be associated with age-related macular degeneration (AMD), but the results were inconsistent. In this study, the cholesteryl ester transfer protein (CETP) rs3764261 variant was significantly associated with an increased risk of AMD (odds ratio [OR] = 1.13, 95% confidence interval [CI]: 1.05–1.21,  Age-related macular degeneration (AMD) is a progressive neurodegenerative disease and the leading cause of irreversible blindness among individuals aged 65 and older, particularly in western countries1The pathogenesis of AMD is still incompletely understood, and a growing body of evidence has suggested that the accumulation of oxidized lipids seems to play a pivotal role in the development of AMD61011Therefore, we conducted a meta-analysis to pool the results of all available studies of CETP rs3764261, LPL rs12678919 and LIPC rs10468017 polymorphisms and the risk of AMD.A total of 165 articles were initially identified from the databases based on our search criteria. After deleting the duplications, 134 articles were left for screening titles and abstracts, with 43 determined to be potentially eligible . After r9101115161718192021222324P > 0.05 for all), except for four studies that did not report the available data. Among the 11 included studies of the LPL rs12678919 polymorphism, seven studies were carried out in Caucasians, and four were performed in Asians. Minor G-allele frequencies ranged from 0.09 to 0.17 in the various populations studied. The average age of subjects ranged from 64.0 years to 81.2 years for cases and from 64.0 years to 77.4 years for controls. There were four GWAS and seven case-control studies. Among the included studies, nine studies adjusted for the CFH gene. In our analysis, no study deviated from HWE in the control groups except for two studies that did not report it. Twelve studies assessed the association between the LIPC rs10468017 polymorphism and AMD. Nine studies were carried out in Caucasians, and three in Asians. Minor T-allele frequencies ranged from 0.11 to 0.35 in different population. Four of the included studies were GWAS. Three studies adjusted for the CFH gene, whereas the others did not adjust for it. The genotype frequency distributions of nine studies were in agreement with HWE, except for the other studies that did not report it.The main characteristics of all included studies are listed in P < 0.05). However, there was not a significant difference between the frequency among Americans and Europeans (P = 0.26). The T allelic frequency in the LPL rs12678919 variant among controls was 10.17% (CI: 8.86–12.66). No significant difference existed in diverse populations . The minor allelic frequency in the LIPC rs10468017 variant among controls was 26.55% (CI: 21.43–31.66), and it was significantly higher in Americans and Europeans than that in Asians . Similarly, no significant difference was detected between Americans and Europeans (P = 0.07).The frequency of the risk allele A in the CETP rs3764261 polymorphism among controls was 30.47% (CI: 26.43–34.52), and it was significantly higher in Americans and Europeans than that in Asians , and this locus was also associated with a significant increased risk of CNV . After adjustment for the CFH gene, the association of the CETP rs3764261 variant with the increased AMD risk remained significant . The result of the stratified analysis by ethnicity revealed that the CETP variant had a significant association with the development of the disease in both Americans  and Europeans  which disappeared in Asians . However, the pooled OR did not show a significant association among the studies without adjusting for the CFH gene . Moreover, no significant association was found when studies had a case-control design excluding GWAS . Moreover, statistically significant associations were noted in elderly individuals. Moreover, we carried out a sensitivity analysis by removing each study one at a time and the pooled ORs remained stable indicating that the results were not influenced by any single study. This analysis also revealed that two studies by Chen et al. were the main source of heterogeneity. After removing these two studies, the I2 statistics for the ORs decreased from 68.5% to 31.2% (P = 0.12), yielding a summary OR of 1.12  with the same direction of effect. We did not find any evidence for the presence of publication bias in the eligible studies .The association between the CETP rs3764261 polymorphism and AMD was investigated in seventeen studies comprising a total of 16888 cases and 15295 controls910111718192021P = 0.17; I2 = 41.9%, P = 0.07). In the subtype analysis for AMD, no significant association was found between the LPL rs12678919 variant and a decreased risk of late-stage AMD . After adjusting for the CFH gene, the LPL locus showed a significant association with increased AMD risk . Subsequently, we also performed stratified analysis by ethnicity, age of case and study design, and no significant associations were found in the other subgroup analysis (P = 1.00) or Egger’s linear regression test (P = 0.49).Eleven studies with a total of 13221 cases and 9611 controls assessed the association between LPL rs12678919 polymorphism and AMD risk910151720analysis . Most ofP < 0.001; I2 = 61.0%, P = 0.02; , P < 0.001; I2 = 51.2%, P = 0.09). When subgroup analysis was conducted for late AMD subtypes, the summary ORs of rs10468017 were 0.80  and 0.74  for CNV and GA, respectively. The stratified analysis revealed that other characteristics including ethnicity, age of case and study design, shared consistency in the direction of effect . After adjusting for the CFH gene, the significant association of the LIPC rs10468017 variant with decreased AMD risk was not appreciably altered . In addition, the pooled ORs remained stable by removing each study one at a time, indicating that the results were not influenced by any single study. There was no evidence for the presence of publication bias (P > 0.05).We subsequently evaluated the relationship between the LIPC rs10468017 polymorphism and the risk of the AMD in twelve studies911151617181920212324For CETP rs3764261, the required information size is 51709 subjects using TSA with the settings mentioned in the Methods section. Before the required information size is reached, the cumulative Z-curve (blue line) has crossed the trial sequential monitoring boundaries (inward sloping red lines), indicating that the positive result was confirmed. This resulting trial sequential analysis was shown in The present meta-analysis showed that the CETP rs3764261 variant was associated with an increased risk of AMD, and the LIPC rs10468017 variant could significantly reduce the susceptibility to AMD. After adjusting for the CFH gene, LPL rs12678919 was also significantly associated with an increased risk of AMD, indicating potential interactions among genes in the complement system and lipid metabolism pathway.et al. and Chen et al. but this finding was contradicted by another GWAS9109Since 2010, several unknown genes associated with AMD have been identified by large-scale GWAS investigations in the high-density lipoprotein cholesterol (HDL) pathway, whereas all previous genetic variants identified from candidate gene testing were mainly found in the complement factor pathway9At a basal state, functional HDL shows anti-inflammatory activity with high levels of antioxidants, active antioxidant proteins, and antioxidant enzymes262829283133After adjusting for the CFH gene, the current study indicates that both the CETP and LPL loci conferred a significantly increased risk of disease, which might contribute to potential interactions among genes in the complement system and lipid metabolism pathway. It has been established that both complement dysregulation and the accumulation of oxidized products of lipid metabolism could modulate genes involving various processes associated with AMD pathology323235In the present meta-analysis, the effect of the CETP rs3764261 variant on AMD might differ in multiethnic populations. In contrast with the absent association in the Asian population, the CETP rs3764261 variant might predispose European and American individuals to develop AMD. It has been observed that the minor allele frequency for this SNP is 0.32 and 0.40 in Europeans and Americans, respectively, which is significantly higher than the frequency of 0.18 observed in Asians. Thus, the consistent effects of these alleles in diverse populations might be due to differences in their genetic backgrounds, suggesting that frequency of the A allele could affect the susceptibility to AMD. However, the different lifestyles in these populations, including smoking, food habits and alcohol consumption, likely also affect the risk of AMD, which might also partly influence our results. Therefore, this finding needs to be considered with thoughtful deliberation, and additional large and carefully designed studies and multivariate analysis that adjusts for confounding factors are necessary.Previous studies have demonstrated that LIPC encodes hepatic triglyceride lipase, which is expressed in the liver2435Several potential limitations should also be acknowledged in interpreting the results from this study. First, as no available cohort studies were found, we could only perform meta-analysis with case-control studies, and this type of study design might be inherently biased by various factors; however, according to the trial sequential analysis, the results of these three SNPs revealed that the power of study was sufficient and the robust evidence might have been confirmed, which adds to the strength of our analysis. Second, both the environmental factors  and other complement factor genes likely affect the risk of AMD, which might also partly influence our results. Our analysis was based primarily on data and information provided from the original literature; however, the included studies did not control for these confounding factors or report sufficient data to analyze the association between CETP/LPL /LIPC genes and AMD adjusted for different environmental factors and other complement factor genes. Thus, the assessment of potential gene-gene or gene-environment interactions was limited, and the possibility that environmental factors and other genes might affect the AMD risk interactively with lipid metabolism could not be excluded in the present study. Further large research studies that allow for the adjustment by these covariates, including genes and environmental factors, should be conducted. Finally, potential publication bias was also a concern. Although we did not observe any apparent evidence of publication bias according to our statistical tests, this analysis does not exclude its possibility. In addition, it was still difficult to fully exclude this problem because there was not a sufficient amount of studies to detect it adequately.In conclusion, the meta-analysis provides evidence for an association between CETP/LPL/LIPC gene polymorphisms and AMD. Carriage of the CETP rs3764261 A allele would increase the risk of developing AMD, and the LIPC rs10468017 variant was associated with a reduced risk of AMD. No significant association between the LPL rs12678919 polymorphism with AMD was observed in diverse populations. Moreover, this study also identified the conceivable interactions between the CETP and LPL loci and the CFH locus in AMD, indicating that CFH may influence the disease risk interactively with lipid metabolism. Future carefully designed studies with large study populations are warranted to substantiate this association and determine these potential gene-gene and gene-environment interactions in the pathogenesis of AMD.A systematic literature search of MEDLINE, EMBASE, and ISI web of science databases through December 2014 was conducted to identify all relevant articles involving the association of the CETP and LPL gene with AMD, using the search terms: (“CETP” or “cholesteryl ester transfer protein” or “LPL” or “lipoprotein lipase” or “LIPC” or “hepatic lipase”) and . The search was not restricted to any language, and we checked the reference lists of retrieved articles and relevant reviews for additional published and unpublished data. When datasets were incomplete for the required data, corresponding authors were contacted for additional information.We performed an initial screen of identified abstracts and titles to examine all retrieved articles. If the studies did not address the association between CETP/LPL /LIPC genes and AMD, they were excluded. Then we further checked the full texts of the remaining articles for their suitability for the present meta-analysis. The full-text articles of all references selected after the application of these criteria were reviewed by using the same criteria. Although cohort, case-control and cross-sectional studies were considered, no cohort or cross-sectional studies were found. For inclusion, studies included in the meta-analysis had to meet the following screening criteria: (1) the primary outcome was clearly defined as AMD; (2) there were at least two comparison groups, e.g., AMD versus control (non-AMD) groups; and (3) odds ratios (ORs) with their 95% confidence intervals (CIs) or sufficient data to calculate these were reported. When multiple publications reported on the same or overlapping data, only the publication with the most updated data was included.P values for Hardy-Weinberg equilibrium (HWE) in controls. If a study provided several risk estimates, the most completely adjusted estimate was extracted. For studies that provided subcategories of AMD disease status, gradings were collapsed into a single AMD group; meanwhile, ORs of early or late AMD (GA or CNV) were also extracted, respectively. The retrieved studies and extracted data from each included study were independently assessed by two investigators (Y.-F.W. and Y.H.). Any inconsistencies were resolved through consensus with a third author (L. M.) for adjudication.From each retrieved study, the following data were extracted: the first author name, the year of publication, study design, country, ethnicity, sample size, the mean age of subjects, diagnosis method, classification criteria, type of macular degeneration, the genotype distributions in cases and controls, the methods for genotyping and Study quality was independently assessed by two reviewers (Y.-F.W. and Y. H.) with the Newcastle-Ottawa quality assessment scale (NOS) which uses a ‘star system’ to evaluate data qualityI2 statistic (I2 > 50% indicated evidence of heterogeneity). Stratified analyses were conducted to explore potential sources of heterogeneity. In addition, we performed sensitivity analysis, after sequentially removing one study at a time, to assess the stability of the pooled results. Potential publication bias was assessed by the application of Egger’s linear regression test, Begg’s rank correlation test and contour-enhanced funnel plots41P value <0.05 was considered statistically significant, except for the tests of heterogeneity, Egger’s linear regression and Begg’s rank correlation in which a level of 0.10 was used.HWE was assessed in the control group of each study via the chi-square test. The strength of the association between the CETP, LPL, LIPC polymorphisms and AMD risk was estimated as OR with CI under an allelic genetic model. In case of significant heterogeneity, random effects models were employed to allow for it, otherwise a fixed-effects model was created. The presence of heterogeneity across individual studies was evaluated by the Q statistic and the According to Cochrane Handbook for systematic reviews of interventions, if all available trials are included, meta-analyses are considered to be the best available evidence. However, a meta-analysis may result in type II errors if data are sparse or may increase type I errors and if there is repeated testing for significance when new trials are added43444543How to cite this article: Wang, Y.-F. et al. CETP/LPL/LIPC gene polymorphisms and susceptibility to age-related macular degeneration. Sci. Rep. 5, 15711; doi: 10.1038/srep15711 (2015).


APOA1) gene play an important role in the regulation of lipids. The objective of the present study was to investigate the effect of two polymorphisms (-75 G/A and +83 C/T) of APOA1 on lipid profiles and the risk of CAD.The disorder of lipid metabolism and genetic predisposition are major risk factors for coronary artery disease (CAD). Variants in the apolipoprotein A1 (APOA1 was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) combined with gel electrophoresis, and then confirmed by direct sequencing.A total number of 300 subjects with CAD and 300 age and sex matched healthy controls were enrolled for the study. Genotyping of the APOA1 -75 AA genotype  and APOA1 -75 A allele  were significantly lower in CAD than in controls. The APOA1 -75 A allele was significantly associated with increasing serum concentrations of ApoA1 and high-density lipoprotein cholesterol (HDL-C) (P < 0.001).The frequencies of APOA1 -75 A allele were likely to have a lower risk of CAD as a result of its effect on higher serum concentrations of ApoA1 and HDL-C.The individuals with the  Coronary artery disease (CAD) is the leading cause of mortality and morbidity in the world . AlthougAPOA1 gene located on the long arm of chromosome 11 [APOA1 gene has been studied extensively [APOA1 gene that involves transition of C-to-T at +83 bp that has also been shown to affect HDL and apoA1 levels [Apolipoprotein A1 (apoA1) is the major apolipoprotein constituent of the high-density lipoprotein (HDL) and is involved in reverse cholesterol transport . Up to nosome 11 . A commoensively . Another1 levels , 19.APOA1 polymorphisms (-75 G/A and +83 C/T) and the risk of CAD, these studies have reported conflicting results [APOA1 on lipid profiles and the risk of CAD in a Chinese population.Although three case-control studies have investigated the association between  results –22. The A total number of 300 subjects with CAD and 300 age and sex matched healthy controls were enrolled for the study between July 2011 and June 2014 in the Second Clinical Medical College of Jinan University, China. CAD cases were defined as those having severe angiostenosis (>50 %) in at least one major coronary artery determined by angiography based on WHO criteria. CAD cases were selected randomly from patients admitted to the hospital and who fulfilled the inclusion criteria. The control group was composed of age and sex matched healthy subjects who had undergone a coronary angiography in the same recruitment period as the CAD patients, with normal ECG, negative stress test, without family history of CAD or other cardiovascular diseases or angiographic evidence of CAD. The study was approved by the Institutional Ethical Committee of the Second Clinical Medical College of Jinan University. A signed informed consent was obtained from all participants in the study. Venous blood was collected in evacuated tubes after an overnight fast of 12 to 14 h.Serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) were measured by the clinical chemistry department using commercial kits following the manufacturer instructions. Serum low-density lipoprotein cholesterol (LDL-C) and very-low-density lipoprotein cholesterol (VLDL-C) were calculated using the Friedwald’s formula. The immunoturbidimetric assay was used to quantify the plasma concentrations of ApoA1 and ApoB.APOA1 gene polymorphisms. Based on the GenBank reference sequence, the PCR primer pair used as follows: forward: 5ʹ-AGG GAC AGA GCT GAT CCT TGA ACT CTT AAG-3ʹ, and reverse: 5ʹ-TTA GGG GAC ACC TAG CCC TCA GGA AGA GCA-3ʹ. The amplified PCR products were digested with 10 units of the restriction endonuclease enzyme MspI overnight. The digested fragments were resolved on a 3 % agarose gel and stained with ethidium bromide for visualization under UV light. The presence of the MspI restriction stie at -75 bp  and at +83 bp  in the 433 bp product resulted in four fragments of 45, 66, 113 and 209 bp. The absence of the restriction site at -75 bp  resulted in three fragments of 45, 179 and 209 bp. The absence of the restriction site at +83 bp  created a larger fragment of 254 bp instead of two fragments of 45 and 209 bp. For quality control, we performed double sampling PCR-RFLP in more than 10 % of the samples and found no differences, and then confirmed by direct sequencing.Total genomic DNA was isolated from peripheral blood leukocytes by the commercially available Qiagen kit . Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay was performed to assess the t test, while those between categorical variables were evaluated using x2 test. The existence of differences in genotypic frequencies between groups was assessed by means of x2 test. The odds ratio (OR) and 95 % confidence intervals (CI) were also calculated. P < 0.05 was required for statistical significance.Statistical analyses were performed by SPSS software for Windows . Allele frequencies were calculated by the gene counting method. Differences between continuous variables were assessed by Student’s P < 0.001), but TG (P < 0.001), TC (P = 0.003), LDL-C (P < 0.001) and VLDL-C (P = 0.004) levels were significantly increased in patients as compared to controls. Multiple logistic regression analysis of known predictors of CAD confirmed the independent role of smoking status (P < 0.001), diabetes (P = 0.001), obesity (P = 0.001), hyperlipidemia (P < 0.001) and ApoA1 (P < 0.001) as risk factors of CAD  and APOA1 -75 A allele  were significantly lower in CAD than in controls  (P < 0.001) (Table The frequencies of 1) Table .Table 2GAPOA1 + 83 C/T polymorphisms and risk of CAD  risk after cardiopulmonary bypass (CPB) surgery. Those patients with the APOA1 -75 AA genotype and A allele had higher 30-day mortality of ALI after CPB surgery [APOA1 -75G/A promoter polymorphism with cognitive performance in multiple sclerosis [APOA1 -75 A allele carriers and breast cancer risk [APOA1 polymorphisms (-75 G/A and +83 C/T) might be susceptibility to myocardial infarction [APOA1 -75 A allele was associated with an increased risk for Alzheimer's disease [APOA1 polymorphisms (-75 G/A and +83 C/T) could be as risk factors for hypertension and obesity [APOA1 -75 G/A polymorphism was associated with gallstone disease and shows sex-specific differences [The  surgery . A case-clerosis . A prospcer risk . A pilotfarction . A case- disease . A Brazi obesity . A case-ferences .APOA1 -75 A allele were likely to have a lower risk of CAD as a result of its effect on higher serum concentrations of ApoA1 and HDL-C, the exact mechanism is still unclear. However, the results have been inconsistent and inconclusive, with few studies reporting either no association or negative association between APOA1 -75 A allele and plasma lipids [Although our results revealed that the individuals with the a lipids –39. Our a lipids , 40–44.Several shortcomings of the present study should be discussed. Firstly, CAD, a multifactorial disease, is influenced by interacting endogenous and exogenous factors, which were not explored in the present study. Secondly, the subjects in this research are only from Han Chinese ethnic group. It would be interesting to conduct similar studies in different populations for comparison. Thirdly, about 50 % cases took statins by which lipid levels are affected. We have adjusted for medicine use in this analysis, but no information could be received on the baseline lipid levels of these cases. Finally, the potential selection bias cannot be avoidable, because this is a hospital based case control study and the subjects may not be representative of the general population.APOA1 -75 A allele were likely to have a lower risk of CAD as a result of its effect on higher serum concentrations of ApoA1 and HDL-C. Additional studies are needed to confirm this finding.In conclusion, our results indicated that the individuals with the


MTHFR) C677T, beta fibrinogen  A455G and T148C, apolipoprotein E (APOE) ε2–4, angiotensin-converting enzyme (ACE) insertion/deletion (I/D), and endothelial nitric oxide synthase (eNOS) G894T. In order to examine these interactions, 712 patients with IS and 774 controls in a Chinese Han population were genotyped using the SNaPshot method, and multifactor dimensionality reduction analysis was used to detect potential interactions among the candidate genes. The results of this study found that ACE I/D and β-FG T148C were significant synergistic contributors to IS. In particular, the ACE DD + β-FG 148CC, ACE DD + β-FG 148CT, and ACE ID + β-FG 148CC genotype combinations resulted in higher risk of IS. After adjusting for potential confounding IS risk factors  using a logistic analysis, a significant correlation between the genotype combinations and IS patients persisted . The results of this study indicate that the ACE I/D and β-FG T148C combination may result in significantly higher risk of IS in this Chinese population.Ischemic stroke (IS) is a multifactorial disorder caused by both genetic and environmental factors. The combined effects of multiple susceptibility genes might result in a higher risk for IS than a single gene. Therefore, we investigated whether interactions among multiple susceptibility genes were associated with an increased risk of IS by evaluating gene polymorphisms identified in previous meta-analyses, including methylenetetrahydrofolate reductase ( Additionally, previous studies have identified susceptibility genes that are involved in the development and progression of IS.Stroke has been recognized as one of the three most common causes of death, and has been established as a major cause of disability in adults. IschemiAlthough numerous studies have investigated potential gene polymorphisms in the pathogenesis of IS, and a large number of candidate genes have been proposed, results from these studies have not always been consistent. The lack of consistency in these association and linkage studies may be due to small sample sizes, or a lack of stroke subtype differentiation. Another intriguing explanation is that the loci may contribute to a certain complex disease only by its interaction with other genes, and the main effects of individual loci may be too small to be observed. As IS hMTHFR) C677T, beta fibrinogen  A455G and T148C, apolipoprotein E (APOE) ε2–4, and angiotensin-converting enzyme (ACE) insertion/deletion (I/D)013) and was conducted according to the guidelines of the Declaration of Helsinki. All participants provided informed consent. If participants were unable to communicate, written consent was obtained from their legal guardians. The ethics committee approved this consent procedure.ACE). The MTHFR C677T, β-FG A455G, β-FG T148C, ApoE ε2–4, and eNOS G894T polymorphisms were genotyped using the SNaPshot method as described previously[5′-CTGGAGACCACTCCCATCCTTTCT-3′ (forward) and 5′-GATGTGGCCATCACATTCGTCAGAT-3′ (reverse). Amplification produced a combination of a 490-bp product and/or a 190-bp product depending on the presence or absence of the ACE insertion allele , respectively. The amplified products were analyzed by electrophoresis on a 1.5% (w/v) agarose gel  and photographed under UV transillumination.Genomic DNA was isolated from a 300-μL blood sample using a TIANamp Blood DNA Kit  according to the manufacturer’s instructions. The extracted genomic DNA was stored at −80°C until genotyping was performed. We investigated six polymorphisms: five were single-nucleotide polymorphisms and one was an I/D polymorphism  and permutation test[http://www.epistasis.org/mdr.html). Single-locus analysis was corrected for multiple testing by using Bonferroni-corrected levels of significance (P′ < 0.008). Other tests were two-tailed and a P-value of 0.05 was considered statistically significant.Alleles were tested for deviation from Hardy–Weinberg equilibrium (HWE) using a chi-square test. A Student tion test. The thrThe baseline characteristics of the participants are listed in All six polymorphisms followed HWE. < 0.0001) and the second greatest CV consistency of 9.0 out of 10. This model was composed of the ACE I/D and β-FG T148C polymorphisms, and was selected as the best model. For the LAA and SAO subtypes, the best model was also a 2-order model composed of the ACE I/D and β-FG T148C polymorphisms . MDR analysis was not carried out for the CE subtype because the sample size available for analysis was too small. MDR analysis was used to detect significant interactions between the candidate genes. According to the results of MDR analysis, we designated the ACE DD + β-FG 148CC, ACE DD + β-FG 148CT, and ACE ID + β-FG 148CC genotype combinations as high risk, and the other genotype combinations as low risk. Logistic regression analysis was used to test the association between the high-risk combinations and risk of stroke. Using the MDR method followed by logistic regression analysis, we showed that the ACE I/D and β-FG T148C polymorphisms were significant synergistic contributors to IS and that the ACE DD + β-FG 148CC, ACE DD + β-FG 148CT, and ACE ID + β-FG 148CC genotype combinations resulted in higher risk for both overall stroke as well as the LAA and SAO subtypes.ACE gene is located on l7q23 and consists of 26 exons and 25 introns. It has been determined that an I/D polymorphism of the ACE gene is related to ACE activity, where the I allele of a 287-bp fragment, and not the deletion allele , is associated with lower ACE activity[The human  activity,15. As e activity. Generat activity. Ang II  activity. Thus, AFGB gene encoding the β-chain is considered the major gene affecting plasma FG levels. A number of previous studies have shown that plasma FG levels may play an important role in the pathogenesis of atherosclerosis[FGB gene polymorphisms are considered a common risk factor for increased plasma FG levels[Fibrinogen (FG) is an important clotting factor and an inflammation marker. FG consists of three pairs of polypeptide chains (Aα/Bβ/Gγ) encoded by the three independent genes for α-FG, β-FG, and γ-FG, respectively. While synthesis of the FG β-chain is the rate-limiting step of FG generation, the sclerosis,20, and FG levels,22. ThusSome studies have shown that interactions among multiple genes may also play a role in the development of IS. A previous study suggesteMany previous studies have examined the association between the ACE I/D or β-FG T148C polymorphism and risk of IS; however, the results were inconsistent. Those genome-wide association studies also did not find an association between the two loci and IS. In our single-locus analysis, no significant associations were found for all polymorphisms, including ACE I/D and β-FG T148C loci. However, our gene-gene interactions analysis found that the ACE I/D and β-FG T148C polymorphisms were significant synergistic contributors to IS. It is possible that the two loci may contribute to IS only by the interaction with each other, and the main effects of individual locus may be too small to be observed.The precise mechanisms by which the gene–gene interactions between ACE I/D and β-FG T148C affect susceptibility to IS are currently unknown, and additional studies are required to clarify how these two genes may increase IS susceptibility. One possible explanation is that both of these two genes are involved in the pathogenesis and severity of atherosclerosis and inflammation, which are two of the principal pathogenetic factors for IS. Another possible explanation is that gene interactions with marginal effects could lead to increased risk of IS via synergistic effects.There are some limitations to our study. First, the study population was limited to southern Han Chinese individuals. Thus, our findings require validation in Han Chinese populations from other regions and in other ethnic populations. Second, the population comprised 1486 individuals in total, which is relatively low, however recruiting >1000 individuals from an ethnically homogeneous population is often sufficient for producing reliable data. Third, the study was cross-sectional, and only suggested a simple association. Future studies should investigate the potential causative relations and underlying mechanism.In conclusion, the present study indicates that the combined ACE I/D and β-FG T148C polymorphisms results in a significantly higher risk of IS, indicating that interactions between these polymorphisms may be synergistically involved in the pathogenesis of IS.S1 TableSNP, single-nucleotide polymorphism.(DOCX)Click here for additional data file.S2 TableSNP, single-nucleotide polymorphism; SR, SNaPshot reverse; SF, SNaPshot forward.(DOCX)Click here for additional data file.S3 Table(XLS)Click here for additional data file.


Caspase-7 (CASP7) may modulate the programmedcell death and thus contribute to cervical cancer risk. In this case-controlstudy of 1,486 cervical cancer cases and 1,301 controls, we investigated associationsbetween four potentially functional polymorphisms in CASP7 and cervicalcancer risk and evaluated their locus-locus interaction effects on the risk.The genotype-phenotype correlation was performed by a generalized linear regressionmodel. We found that the rs4353229 polymorphism was associated with cervicalcancer risk .Compared with the TT genotype, the rs10787498GT genotype was associated withan increased cervical cancer risk .Combination analysis showed that subjects with four putative risk genotypeshad a 1.54-fold increased cancer risk, compared with those who carried threeor less putative risk genotypes. We also observed significant locus-locusjoint effects on the risk, which may be mediated by the polymorphisms regulating CASP7mRNA expression. Subsequent multifactor dimensionality reduction and classificationand regression tree analyses indicated that the CASP7 genotypes mighthave a locus-locus interaction effect that modulated cervical cancer risk.Out data suggest that CASP7 polymorphisms may interact to modify cervicalcancer risk by a possible mechanism of regulating CASP7 mRNA expression.Polymorphisms in  Cervical cancer is one of the leading cancers in women worldwide, with529,800 new-diagnosed cancer cases and 275,100 cancer deaths in 200814Caspase-7 (CASP7) gene may modulate the default programmedcell death, thus leading to genomic instability and contributing to inter-individualvariation in cancer susceptibility12Caspases, at the heart of the apoptotic machinery, encode an evolutionaryconserved family of cysteine-aspartic acid proteases and coordinate in cellularregulation and execution of apoptosisCASP7SNPs and cervical cancer risk, besides genome-wide association studies. Herein,we performed a relatively large case-control study to test the hypothesisthat potentially functional SNPs in the CASP7 3′-UTR are independentlyand/or jointly associated with cervical cancer risk.To date, no published studies have investigated associations between functional P = 0.126) as result ofmatching. However, the differences in age at primiparity, menopausal statusand body mass index (BMI) were significant between cases and controls. Therefore,we subsequently adjusted these variables for any residual confounding effectin multivariate logistic regression analyses.The selected characteristics of the study subjects are listed in CASP7SNPs . We then explored their two-locus joint effects. As shown in Pfor homogeneity = 0.016 and 0.007; respectively). Although no difference inrisk estimates by the homogeneity tests, we observed a highly associated riskfor the presence of rs10787498GT/GG and rs4353229TT genotypes  as well as a board-line significance forthe joint effect of rs12247479AG/AA with rs4353229TT genotypes and for thatof rs10787498G with/without rs12247479A allele .In further logistic regression analyses, we observed a significant locus-locusmultiplicative interaction between rs1127687 and rs12247479 as well as betweenrs1127687 and rs10787498  values forall observed significant associations . Thers4We further performed the multifactor dimensionality reduction (MDR) analysisand found that rs4353229 was the best one-factor model with the highest cross-validationconsistency (CVC) (89%) and the lowest prediction error (48.0%) among allfour SNPs. Additionally, the four-locus model had a maximal CVC (100%) anda minimal prediction error (46.8%), suggesting a better prediction than othermodels . SubsequP = 0.041; By the classification and regression tree (CART) analysis, we found rs4353229to be the initial split of root nodes, indicating that rs4353229 was the strongestrisk factor for cervical cancer among these four SNPs. Further inspectionof the tree structure revealed distinct interaction patterns. Women carryingrs4353229TT, rs12247479GG, rs10787498GT/GG and rs1127687AG/AA genotypes [terminalnode (TN) 6] had a 1.67-fold increased risk of cervical cancer, comparedwith the TN3 group at the lowest risk , P = 0.056; CASP7 mRNA expression levels showed an increased trend forrs10787498TT-rs12247479GG carriers and a decreased trend for rs1127687AG/AA-rs10787498GT/GGcarriers was 0.034 for rs10787498, but given that the deviation from HWE among controlswas defined as a significance level of α <10−3or 10−4, all the SNPs in our analyses were in agreementwith HWE. Finally, because the lack of routine HPV screening for all casesand controls in our hospital, we could not evaluate HPV infection as the potentialconfounder in risk estimates of cervical cancer.Despite the strengths and biologic plausibility of the associations observedin the current study, several limitations need to be addressed. Firstly, theremay be selection and information bias originated from a retrospective studydesign, which may have been minimized by frequency-matching for cases andcontrols as well as the adjustment for potential confounding factors in multivariateanalyses. Secondly, the CASP7 SNPs might be associated with cervicalcancer risk in Eastern Chinese women. There were substantial joint effectsand locus-locus interactions among these SNPs, and such effects may contributeto cervical cancer risk by affecting CASP7 mRNA expression. However,well-designed, larger, and prospective studies with detailed information aboutHPV infection are warranted to validate our findings.In summary, in the current case-control study of 1,486 cases and 1,301controls, we found that The recruitment of the cases and controls was partly described previouslyhttp://www.ncbi.nlm.nih.gov/projects/SNP) and the International HapMap Project database (http://hapmap.ncbi.nlm.nih.gov/),we found that there were 22 SNPs in CASP7 3′-UTR, of which fourwere finally selected for genotyping, based on the following criteria: 1)minor allele frequency of at least 5% in Chinese populations, 2) with lowlinkage disequilibrium by using an r2 threshold of <0.8for each other, 3) predicted to be potentially functional by the SNP functionprediction platform (http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm),and 4) not included and published in genome-wide association studies. Thus,the selected SNPs were rs4353229 T> C, rs12247479 G> A, rs10787498 T>G and rs1127687 G> A. Genomic DNA extraction and genotyping were conductedas described previouslyBy searching the NCBI dbSNP database (CASP7genotypes and CASP7 mRNA expression levels both available for 270 HapMapsubjects by SNPexp online tool (http://app3.titan.uio.no/biotools/help.php?app=snpexp) and conducted genotype-phenotype correlation analysis as described previously18To evaluate biological plausibility of our findings, we used the data on 2-test for each SNP. We performedthe Pearson's χ2-test for the differences in selectedvariables between cases and controls. The association of CASP7 genotypeswith cervical cancer risk was estimated by computing ORs and their 95% CIsfrom both univariate and multivariate logistic regression models. We alsoevaluated the associations in subgroup and joint effect analyses. The PROCHAPLOTYPE procedure in SAS software was applied to infer haplotype frequenciesamong the four SNPs. To avoid false positive associations in this study, wecalculated the FPRP with the assumption of different prior probabilities . FPRP values <0.2 were considered to be noteworthyt test and analysis of variance test to evaluate thedifferences in the relative mRNA expression levels among different genotypegroups.HWE was tested by χhttp://www.multifactordimensionalityreduction.org/)and SAS software , respectively, asdescribed previouslyn-factor interactionmodel. Then, we performed the interaction dendrograms and graphsThe MDR and CART analyses were conducted by the MDR V2.0 beta 8.2 program, unless stated otherwise. All All authors contributed significantly to this work. Conceived and designedthe study strategy: X.C. & Q.W. Designed the experiment: Q.W. & T.-Y.S.Recruited the participants and collected their information and blood samples:T.-Y.S., J.H., K.-D.Y., Z.-M.S., M.-H.S. & X.W. Performed the experiments:T.-Y.S., M.-Y.W. & M.-L.Z. Statistical analyses: T.-Y.S. & J.H. Wrotethe manuscript: T.-Y.S. & Q.W. All authors reviewed the manuscript. Inaddition, all authors approved the final draft.Supplementary Information


In a recent study, rich clinical assessment and longitudinal study design are combined with host gene expression and microbial sequencing analyses to develop a framework for exploring disease etiology and outcomes in the context of human inflammatory disease.http://dx.doi.org/10.1186/s13059-015-0637-xSee related article:  Modulating the microbiome through prebiotics, probiotics and antibiotics holds tremendous potential for the treatment and prevention of human inflammatory disorders, but there is a need for medical research to explore how alterations in microbial communities are associated with disease state and host physiology. Few diseases have an easily explained etiology as outcomes are affected by a myriad of factors, including host genetics, microbial communities and environmental factors. Well-designed longitudinal human clinical studies are necessary to move from ‘correlation’ to ‘causation’. However, integrating rich clinical metadata with large datasets characterizing microbial and host attributes is a daunting task. A recent article by Morgan, Kabakchiev and colleagues lays out the analytic framework necessary to achieve this goal .et al. analyzed samples from ulcerative colitis (UC) and familial adenomatous polyposis (FAP) patients who had undergone ileal pouch-anal anastomosis (IPAA) surgery or so-called ‘J-pouch’ construction. Genetically, the auto-inflammatory nature of UC is associated with polymorphisms in 160 genes, whereas FAP displays a more classic Mendelian inheritance linked to mutations in a single gene [In their study, a complex set of interwoven diseases and outcomes is explored simultaneously to tease out the contributions to clinical outcome of several factors: underlying disease, host gene expression and mucosal microbiome composition . Morgan gle gene . Outcomegle gene . This clgle gene . With knPrevious studies have looked at microbial communities and host transcriptomes in patients who have undergone IPAA; however, thus far none has studied them simultaneously to explore host-microbe interactions. In their study, Morgan and colleagues measured host gene expression and microbial community composition in paired biopsies (pouch/afferent limb) from 265 patients undergoing IPAA because of UC or FAP . They uset al. elegantly explain how, given their sample size and the desire to retain 90% power and an alpha equal to 0.05, thousands of variables must be reduced to approximately 100 transcripts and 100 bacterial clades. From these 104 pairwise tests, statistically significant conclusions between the datasets could be drawn.Identifying associations between datasets of this size requires an unreasonably large sample size or the compression of many variables into a few. The required size of the population or the degree of compression can be calculated with power equations. In their study, Morgan To achieve the data compression required by the power equation, Morgan and colleagues employed various dimension-reduction techniques . The issThe microbial OTUs were first filtered for abundance and then subjected to unsupervised and supervised cluster analyses. For unsupervised dimensionality reduction, OTUs were transformed into nine clade principal components (cPCs), which explained 50% of the observed variance . For supEscherichia and Actinobacteria, which are positively and negatively associated with inflammation, respectively.In addition to transcriptome and microbiome data, extensive metadata, including antibiotic usage, score of inflammation and post-surgical outcomes, were collected for each patient . To uncoBacteroides and Firmicutes. Overall, this modest number of interactions indicates that microbial composition is more likely shaped by early-life colonization or diet than by local gene expression [The novelty of this research is the incredibly rich, well-performed clinical study and the depth of analyses focused on host-microbe interactions. Such comparisons would not have been possible without the supervised and unsupervised data reduction methods. Using multivariant linear modeling to control for antibiotics, inflammation and outcome, Morgan and colleagues measured gene-clade associations . Surprispression .Next, Morgan and colleagues applied the gene-medoids and representative clades from supervised clustering to linear discriminant analysis (LDA) . They waThe dominant effect of antibiotic usage was a common theme throughout this paper. Antibiotics were the main driver of microbial community composition and highly predictive of the chronic pouchitis outcome, which is unsurprising given that antibiotics are often prescribed to treat pouchitis . AntibioClostridium difficile colonization-resistance studies in humans could be investigated with a mouse model [C. difficile. Ideally, these results will soon be brought back to humans and tested in a controlled clinical setting. Clinical studies are essential to generate hypotheses about what should be modeled and tested in animals and then to validate concepts derived from mouse studies in an iterative fashion.A common goal of clinical studies is to better stratify and predict disease outcomes. In this particular study, that goal was not realized owing to complications imposed by the patients’ antibiotic usage history and lack of multiple time-points - factors that limit many clinical studies. For human treatment studies, many factors confounding analysis cannot be controlled without placing an undue burden on the patient - that is, asking patients to provide frequent biopsies or to refrain from taking antibiotics. Thus, when available, animal models of a disease can be useful for well-controlled explorations of hypotheses generated from clinical studies. A recent paper by Buffie and colleagues elegantly demonstrated how hypotheses from se model . This huMorgan and colleagues used 16S amplicon sequencing and microarrays to infer host-microbe interactions [