MTHFR) polymorphisms and acute myeloid leukemia risk (AML) have yielded inconsistent results. The aim of this study is to derive a more precise estimation of the association between MTHFR (C677T and A1298C) polymorphisms and acute myeloid leukemia risk. PubMed and Embase databases were systematically searched to identify relevant studies from their inception to August 2013. Odds ratios (ORs) with 95% confidence intervals (CIs) were the metric of choice. Thirteen studies were selected for C677T polymorphism (1838 cases and 5318 controls) and 9 studies (1335 patients and 4295 controls) for A1298C polymorphism. Overall, pooled results showed that C677T polymorphism was not significant associated with AML risk. Similar results were observed for the A1298C polymorphism and in subgroup analysis. All comparisons revealed no substantial heterogeneity nor did we detect evidence of publication bias. In summary, this meta-analysis provides evidence that MTHFR polymorphisms were not associated with AML risk. Further investigations are needed to offer better insight into the role of these polymorphisms in AML carcinogenesis.Previous observational studies investigating the association between methylenetetrahydrofolate reductase ( Worldwide, an estimated 57 000 cases of leukemia occur every year MTHFR) gene is found at the end of the short arm of chromosome one at locus 1p36.3. The complementary DNA sequence of this gene is approximately 2.2 kb, made up of 11exons (103–432 bp). The major product of MTHFR locus in human is a 77-kilodaltonprotein MTHFR plays a pivotal role in the folate metabolism, it can catalyze the irreversible conversion of 5, 10-methylenetrahydrofolate to 5-methylenetrahydrofolate, which participates in the remethylation of homocysteine to methionine MTHFR, C677T and A1298C, have been associated with reduced enzyme activity of MTHFR, which lead to an accumulation of 5, 10-methylenetetrahydrofolate and DNA hypomethylation. The 5,10-methylenetetrahydrofolate donates a methyl group, which converts dUMP to dTMP and repairs DNA damages MTHFR 677TT genotype have 30 percent enzyme activity compared with those having wild-type allele, while those with heterozygous MTHFR 677 CT allele have 60 percent enzyme activity MTHFR gene is A1298C in exon7, which leads to a glutamate-to-alanine (A>C) change and reduced enzyme activity of MTHFR.The folate metabolites of carcinogens can influence the gene expression and DNA instability. DNA translocations, inversions or deletions in haematopoietic progenitor cells will lead to leukemia. Be short of folate can result in a lot of cellular disorders MTHFR polymorphisms and AML risk MTHFR polymorphisms and AML risk.To date, several studies have investigated the association between MTHFR, polymorphism, acute myeloid leukemia or acute myeloblastic leukemia. We did not apply language restrictions. References of reviews or original studies identified in the literature search were hand searched for additional studies. Studies were included if they met the following inclusion criteria: (1) explored the association of MTHFR (C677T and A1298C) polymorphisms with AML risk; (2) used a case-control design; (3) provided available genotype or allele frequency of the cases and control to calculate ORs with 95% CIs. The exclusion criteria also applied: the data from study were repeated or overlapped; there was no available genotype or allele frequency; the patients were about therapy-related AML; the studies were review, case report, or comment.Eligible studies were identified by searching electronic literature databases PubMed and Embase (from inception to August 2013). The search strategy used the following keywords: MTHFR C677T and A1298C. Any disagreements were resolved by consensus and a third author (YZ). All data were extracted from the published studies and no authors were contacted to require further information.Two investigators (YTQ and FW) independently extracted data using a standardized data-collection form. Study characteristics extracted from each article were as follows: first author, year of publication, country of origin, racial decent, participant age, number of participants, source of controls, genotype studied, and available genotype frequency information for MTHFR (C677T and A1298C) polymorphisms and AML risk was measured by using crude odds ratio (OR) with 95% confidence interval (CI). The pooled ORs were estimated in following models: allele contrast (T vs. C), codominant model , dominant model (TT+CT vs. CC), and recessive model (TT vs. CT+CC), respectively. For MTHFR A1298C polymorphism, we assessed the same association. The Cochran Q test was used to test statistical heterogeneity. The I2 statistics P value of less than 0.1 for the Q statistic was considered as heterogeneity across studies, allowing for the use of a random-effects model . MTHFR A1298C polymorphism and AML risk. Overall, the estimate results indicated non-significant increased risk association of MTHFR A1298C polymorphism with AML risk in some genetic models , without zero heterogeneity . The Begg rank correlation test and Egger linear regression tests for publication bias in the meta-analysis indicated no obvious publication bias among studies  and 9 studies (1335 patients and 4295 controls) explored the association between the C677T and A1298C polymorphisms and AML risk, respectively. Results of this study suggested that MTHFR (C677T and A1298C) polymorphisms were not significantly associated with AML risk. Moreover, similar results were observed in subgroup analyses based on ethnicity, sample size, and HWE in controls.To the best of our knowledge, this is the first meta-analysis to assess the association between MTHFR (C677T and A1298C) polymorphisms and risk of several cancers. For instance, You et al have demonstrated that the MTHFR C677T and A1298C polymorphisms were associated with bladder cancer risk MTHFR C677T polymorphism increased the risk for developing colorectal cancer MTHFR C677T polymorphism and susceptibility to ovarian cancer MTHFR A1298C polymorphism head and neck cancer Nowadays, several meta-analyses have been performed to clarify the association between MTHFR 677 TT genotype, lack of vitamins B6 and B12, methionine and folate, and high consumption of alcohol are at increased risk of developing colorectal tumors THF, so lack of folate can intervene thymidylate biosynthesis and then lead to leads to errors in DNA synthesis, strand breakage, and chromosomal repair. (2) Low-level 5-methylTHF may result in DNA hypomethylation and cause proto-oncogene expression due to cellular S-adenosylmethionine used up. Thus, cohort studies are needed to focus on gene-nutrient interactions in the future.Several studies have demonstrated that individuals with MTHFR (C677T and A1298C) polymorphisms and AML risk were found in sample size subgroups or all in HWE, which indicated that the results of our analysis was reliable and stable. The real effect of MTHFR (C677T and A1298C) polymorphisms may be concealed by the causal genes in AML. Moreover, different ethnicity of genotypic milieu and living surroundings might have an effect on AML risk, which may led to an effect in our results.In order to better estimate the association of MTHFR (C677T and A1298C) polymorphisms with AML risk, subgroup analysis based on ethnicity, sample size and HWE, was performed. Although Asian and Brazilian subgroups were at increased risk in some genetic models, no significant associations between MTHFR (C677T and A1298C) polymorphisms on AML risk. Third, there were no significant effects for both polymorphisms. Fourth, gene-gene and gene-environment interactions might also be considered in future studies. In spite of these, our meta-analysis also has two advantages as follows: (1) there was no significant absence of evidence of publication bias in the present study, which highlighted further, ensured the reliability of association analysis our findings. (2) There was no evidence of statistical heterogeneity between the analyses of two polymorphisms and AML risk underpins the combinability of the component studies.Several limitations might be acknowledged in this meta-analysis. First, we only selected the published articles to acquire data for analyses, and the unpublished article’s effect was unknown. Thus, it is necessary to conduct a system review to avoid the potential effect in analysis. Second, our study was based on single-factor estimate, which explained the effects of two polymorphisms on AML risk respectively and lack of combination of two polymorphisms analysis. So, conducting a meta-analysis to investigate the combination of these two functional polymorphisms may offer better insight into MTHFR C677T polymorphism is not associated with AML risk, as well as A1298C polymorphism. Future well-design study is warranted to estimate the effect of combination of two polymorphisms and gene-environment interactions. If epidemiologic study confirms the role of gene-environment interactions, additional studies will be needed to further elucidate the potential biological mechanisms involved.In conclusion, our meta-analysis indicates that Figure S1Flow chart.(DOC)Click here for additional data file.Checklist S1PRISMA checklist.(DOC)Click here for additional data file.


Childhood sexual abuse (CSA) is a traumatic life event associated with an increased lifetime risk for psychopathology/morbidity. The long-term biological consequences of CSA-elicited stress on chromosomal stability in adults are unknown. The primary aim of this study was to determine if the rate of acquired chromosomal changes, measured using the cytokinesis-block micronucleus assay on stimulated peripheral blood lymphocytes, differs in adult female monozygotic twins discordant for CSA.Monozygotic twin pairs discordant for CSA were identified from a larger population-based sample of female adult twins for whom the experience of CSA was assessed by self-report . Micronuclei (MN) contain chromatin from structurally normal or abnormal chromosomes that are excluded from the daughter nuclei during cell division and serve as a biomarker to assess acquired chromosomal instability.t-test, t16 = 2.65, P = 0.017). No additional effects of familial factors were detected after controlling for the effect of CSA exposure. A significant interaction between CSA history and age was observed, suggesting that the biological effects of CSA on MN formation may be cumulative.Female twins exposed to CSA exhibited a 1.63-fold average increase in their frequency of MN compared to their nonexposed genetically identical cotwins (Paired These data support a direct link between CSA exposure and MN formation measured in adults that is not attributable to genetic or environmental factors shared by siblings. Further research is warranted to understand the biological basis for the observed increase in acquired chromosomal findings in people exposed to CSA and to determine if acquired somatic chromosomal abnormalities/somatic clonal mosaicism might mediate the adult pathology associated with CSA. Childhood sexual abuse (CSA) not only compromises well-being in childhood but is also associated with a broad range of psychopathology and morbidity in adulthood Given that both telomere shortening and/or inflammation-related generation of oxygen and/or nitrogen species are phenomena that have been associated with an increased frequency of acquired chromosomal abnormalities The cytokinesis-block micronucleus (CBMN) assay is an attractive biomarker for estimating chromosomal damage associated with environmental insults or exposures and is considered an acceptable alternative to data obtained from the assessment of metaphase chromosomal analyses MN frequency is influenced by both heritable genetic and environmental factors In the present study we tested the hypothesis that adult females who experienced CSA have a higher frequency of spontaneously occurring leukocyte MN than their identical twins who did not experience CSA. Although these twin pairs are quite rare, we elected to use a discordant identical twin study design since it allows one to control for known genetic influences on MN formation Human subjects research was approved by the Virginia Commonwealth University IRB (#12407 and #179). Written informed consent was obtained from all research participants.Female-female CSA discordant MZ twins were ascertained from the population based Virginia Adult Twin Study of Psychiatric and Substance Use Disorders “Before you were 16, did any adult, or any other person older than yourself, involve you in any unwanted incidents like (i) inviting or requesting you to do something sexual, (ii) kissing or hugging you in a sexual way, (iii) touching or fondling your private parts, (iv) showing their sex organs to you, (v) making you touch them in a sexual way or (vi) attempting or having sexual intercourse?”non-genital (N = 3 pairs) , (2) genital (N = 8 pairs) [numbers (iii) and (v)] and (3) intercourse (N = 6 pairs) [number (vi)]. The mean age at the time of the first CSA incident was 10.7 years-old (s.d. = 3.9) and ranged from 5 to 16 years of age. These CSA discordant MZ twin pairs were invited to complete a health history questionnaire and submit blood samples (VCU IRB #12407). After providing their informed consent, blood samples were obtained by a health care provider of the participants’ choosing and shipped to our cytogenetics laboratory (overnight delivery carrier) at room temperature per standard procedures. A random sample of age-matched female MZ twins  was also obtained from the MATR to serve as an unselected reference sample (VCU IRB #179), with health history questionnaire completion and blood specimen processing occurring using the identical protocol. The mean current age of the discordant MZ twin pairs  was not significantly different from that of the reference sample  .Of the 1411 individuals completing this portion of the interview, there were 326 MZ twin pairs where both twins provided information about CSA exposure, of which 74 pairs were classified as discordant for CSA. For the present study, specimens were collected from 17 of these discordant MZ twin pairs  in whichA number of psychiatric and substance use disorders were assessed multiple times in the discordant twins using DSM-IIIR Twin zygosity status was confirmed, using genomic DNA that was isolated from whole blood using the Puregene DNA Isolation Kit (Qiagen), based on the patterns of 13 highly polymorphic short tandem repeat sequences .To ensure that erythrocytes did not confound the recognition and scoring of MN, leukocytes were isolated using Histopaque-1077 (Sigma) and then established in culture according to standard procedures  MN were visualized following Giemsa staining (4% Harleco Giemsa solution) and identified according to the criteria established by Fenech et al. t-test. A general effect of CSA, not differentiated by severity of exposure, was examined in all tests since this was deemed appropriate based on the current literature t-test since data transformations were not required and it provided an additional safeguard against biases sometimes encountered with modest sample sizes. Two-sided P-values were reported for all pairwise comparisons and exact P-values were calculated for non-parametric tests.Differences in MN rates and NDCI values in the CSA exposed twins versus their nonexposed cotwins were assessed by a paired Student’s One could speculate that the nonexposed twin of pairs discordant for CSA could have elevated MN levels because of exposure to other shared adverse family factors not directly related to CSA or potentially from stress arising from knowledge that her cotwin was abused. To further test whether an effect of CSA on MN levels was restricted only to the abused twin a population sample of age-matched MZ female twins was incorporated in the analyses to serve as a reference group. Tests were performed using generalized mixed-effect models t-test, but showed no significant difference   and resulted in a significant Wilcoxon rank sum test . We then tested if these results were largely influenced by the discordant twin pair having the largest difference in MN level . CSA discordant cotwins did not differ on measured diet and lifestyle factors and no significant differences were found for rates of adult disease  . In cont disease . It shouP<0.001), there was no indication of an additional effect attributable to the familial environment (P = 0.406) based on results from generalized mixed-effect models. Removal of the most extreme value in the reference sample (greater than 3 standard deviations from the mean) resulted in a reduction of the mean MN level to12.3 and a more similar estimate of variability as the CSA nonexposed sample (SD = 5.4), but still yielded similar modeling results as the full sample.The overall mean [SD] MN frequency in CSA exposed twins was 22.0 [11.3] compared to 14.9 [5.6] per 1000 cells in their nonexposed cotwins . The meaP = 0.661), since the twins selected for study were from a narrow age range. However, the difference in MN frequencies between discordant cotwins showed a significant interaction with age, with the divergence increasing over time  (P = 0.001) rather than a decrease in MN level for the CSA nonexposed group . There was no increase in MN frequency with age over the limited age-matched range evaluated for the reference group (P = 0.361).Not surprisingly, the clearly established association of age with MN that has been shown by our lab and several previous investigators was not identified in this study, as estimated using mixed-effect models ( 0.0006) . SeparatThe present study yielded three main findings. Firstly, female twins exposed to CSA had an increased frequency of acquired somatic chromosomal changes (measured using MN), compared to their nonexposed, genetically identical (MZ) cotwins. Secondly, our analyses ruled out the hypothesis that the higher MN level was due to an indirect association through other shared familial risk factors. Thirdly, evidence was found for an increase in MN level with age in the CSA exposed twins that was not observed in their genetically identical nonexposed cotwins. This latter finding suggests that there may be a cumulative effect of CSA exposure on the frequency of acquired MN in lymphocytes.in vitro induction (primarily using 5-azacytidine); or (2) as a result of mutation . The results of these investigation have shown increases in the rate of micronuclei associated with methylation alterations , with observed delays in centromere separation being suggested as at least one means whereby the observed increase in somatic chromosomal abnormalities was acquired The short- and long-term negative sequelae of CSA have been extensively documented. Kendler, et al. Interestingly, we observed a significant statistical interaction whereby only twins exposed to CSA displayed an increased MN level over the limited age range evaluated in this study. This association suggests that these individuals accumulated chromosomal changes over their lifespan, with this increase appearing to be in addition to normal, age-related or stochastic events. It is unlikely that this increase was limited to an effect of the normal aging process In this study, the relationship between CSA and MN formation was tested using a powerful model; the discordant MZ twin design. Perhaps the most significant advantage of this approach is its ability to address issues related to direction of causality within cross-sectional data. Given that CSA has been shown to correlate with multiple family background risk factors, nearly all of which are shared by twins , the discordant MZ twin design was effective for controlling for the effect of these influences. Without sufficient control in epidemiological samples, which necessitates the measurement of all confounding factors (some of which are unknown) the clustering of childhood adversities would likely serve to overestimate the association between CSA and MN formation. Similarly, the use of MZ discordant twins served as a control for factors related to MN formation that could be shared by twins, including but not limited to, inherited defects in genome maintenance Although our discordant MZ twin study design provided a powerful test of our primary hypothesis, this investigation had methodological limitations. For instance, since we studied lymphocytes, the observation of an increased frequency of acquired chromosomal changes in twins experiencing CSA might have the greatest relevance to health problems associated with the cascade of biological effects mediated through the inflammatory system and may or may not be directly applicable to the acquisition of adult psychopathology. Indeed, this tissue sampling limitation may explain, at least in part, the lack of a clear relationship between MN frequency and the presence of adult psychopathology/morbidity for the limited number of conditions evaluated in our sample. However, it is of interest to note that chromosomal changes, primarily aneuploidy, are acquired in many tissues, normal brain and nerve cells, throughout development and that the brain and other somatic cells from individuals having a variety of health and/or psychiatric conditions show higher levels of acquired chromosomal abnormalities than controls In summary, our study results showed increases in acquired chromosomal instability in female twins exposed to CSA, with the effect appearing to be cumulative with age, and independent of the family environment. Improvements in our understanding of this and other biological changes associated with CSA could lead to the development of biomarker panels for identifying individuals who are most at risk for acquiring health problems. In addition, these persisting biological alterations underscore the gravity of sexual abuse in children.


Corydalis yanhusuo alkaloid (CA) is a major component of Qizhiweitong (QZWT) prescription which has been used for treating gastric ulcer for centuries and its mechanism remains unclear completely. Metabolite profiling was performed by high-performance liquid chromatography combined with time-of-flight mass spectrometry (HPLC/ESI-TOF-MS) and in conjunction with multivariate data analysis and pathway analysis. The statistic software Mass Profiller Prossional (MPP) and statistic method including ANOVA and principal component analysis (PCA) were used for discovering novel potential biomarkers to clarify mechanism of CA in treating acid injected rats with gastric ulcer. The changes in metabolic profiling were restored to their base-line values after CA treatment according to the PCA score plots. Ten different potential biomarkers and seven key metabolic pathways contributing to the treatment of gastric ulcer were discovered and identified. Among the pathways, sphingophospholipid metabolism and fatty acid metabolism related network were acutely perturbed. Quantitative real time polymerase chain reaction (RT-PCR) analysis were performed to evaluate the expression of genes related to the two pathways for verifying the above results. The results show that changed biomarkers and pathways may provide evidence to insight into drug action mechanisms and enable us to increase research productivity toward metabolomics drug discovery.Metabolomics, the systematic analysis of potential metabolites in a biological specimen, has been increasingly applied to discovering biomarkers, identifying perturbed pathways, measuring therapeutic targets, and discovering new drugs. By analyzing and verifying the significant difference in metabolic profiles and changes of metabolite biomarkers, metabolomics enables us to better understand substance metabolic pathways which can clarify the mechanism of Traditional Chinese Medicines (TCM).  Helicobacter pylori, non-steroidal anti-inflammatory drug use (NSAIDs), alcohol and infection Gastric ulcer is a widespread disease that afflicts many people all over the world due to its higher and higher morbidity. According to the statistics of 2005, the incidence of gastric ulcer was up to 80%, especially western world. It has 40–80% of recurrent frequency all over the world. Gastric ulcer in humans occur frequently because of various endogenous and exogenous factors such as stress, smoking, nutritional deficiencies, hydrochloric acid, pepsin, Corydalis yanhusuo, Radix Glycyrrhizae and Radix Bupleuri ect. have been extensively used to cure gastric ulcer for centuries in China, owing to its significant therapeutic performance in clinical application Corydalis yanhusuo have been investigated in our previous study. Tetrahydropalmatine, corydaline, protopine et al were the biological activities of Corydalis yanhusuo. The structures of the constituents were examined. CA is acknowledged to be the major active component in Corydalis yanhusuoQZWT prescription composed of To explain the action mechanism of drugs, metabolomics methodology has been widely used Recent advances of instrumentation and computation have enabled the simultaneous analysis of a large number of metabolites. HPLC coupled with MS has been proven to be an effective combination for metabolites identifications and quantifications due to its excellent resolution and sensitivity. The aim of current study was to obtain a systematic view to dissect the mechanism of CA as an effective treatment for gastric ulcer. The specific and unique biochemical pathways of drug efficacy can be identified, when coupled with multivariate data analysis techniques. The purpose of this study is to identify multiple metabolites that could facilitate the understanding of the action mechanism of CA and aid their incorporation into future improvement of TCM therapy.All experiments were performed in accordance with the approved animal protocols and guidelines established by Medicine Ethics Review Committee for animal experiments of Liaoning University of Traditional Chinese Medicine.Seven-week-old male SD rats weighing 200–250 g, were provided by the experimental animal centre of Dalian Medical University. The care and handling of rats were in accordance with the standard of Specific Pathogen Free. Gastric ulcer was induced in the rats according to the method in a previous report with a slight modification On the last day, rats were deeply anesthetized and then sacrificed. Blood was collected, plasma and serum were separated via centrifugation at 3000 rpm for 15 min at 4°C. The plasma samples were collected and stored at −80°C flash frozen in liquid nitrogen until metabolomics analysis were performed. Then, the stomachs were cut along the greater curvature, washed with saline. The area of ulcer was measured by a compass to measure ulcer index. The area of ulcer equals to the width of the ulcer times the length of the ulcer. For histological evaluation, gastric tissue samples were fixed in neutral buffered formalin for 24 h. Stomach sections were dehydrated with graded ethanol, passed through xylene, and embedded in paraffin. Paraffin sections (5 mm thickness) were stained with hematoxylin/eosin (HE). The other gastric ulcerated tissues were rapidly removed and frozen in liquid nitrogen until the extraction of total tissue RNA.Chromatography was performed using an Agilent 1100 series HPLC system equipped with quaternary pump, online degasser, autosampler, and thermostated column compartment. The injection volume was fixed at 4 µL. All the samples were maintained at 4°C during the analysis. The separation was performed on a 4.6*100 mm, ZORBAX SB-C18 column . The column temperature was set at 45°C. The mobile phases were composed of 0.1% formic acid in water (solvent B) and 0.1% formic acid in acetonitrile (solvent A), the flow rate was set as 1 ml/min with split ratio 1∶3, the gradient was used as follows: a linear gradient of 70– 33% B over initial– 5.0 min, 33 –98% B over 5.0–12.0 min. The eluent was introduced to the mass spectrometer directly. After every 10 samples injecting, a pooled sample as the QC sample followed by a blank was injected in order to ensure the stability and repeatability of the LC-MS systems.For mass spectrometry, the Agilent 6220 TOF-MS with an electrospray ionization source (ESI) in negative mode was used. The flow rate of dying gas (N2) was set at 9 L/min. The nebulizer was set at 45 psi. The other optimal conditions were as follows: dying gas temperature of 350°C, fragment voltage of 120 V. Data were collected in the full-scan mode from m/z 50 to 1050 amu over 0–12 min. The MS data were collected in centroid mode.Data analysis procedure is shown in The identification of potential biomarkers was determined by Q-TOF (Xevo G2). The MS collision energy is 35ev, and the data was obtained in the negative ion mode, x (V4. MassLn1) software was used for data analysis. The identities of the specific metabolites were confirmed by elements information comparison of their mass spectra using the elemental composition information provided by the software.MPP software was employed to all significant (folder change >2) up regulated and down regulated metabolites and related biological pathways. The potential markers identified were compared with the accurate mass charge ratio in some databases, including HMDB, KEGG, METLIN, LIPID MAPS and PUBCHEM, to discover related pathways. T-test and fold-alter were used to determine the statistical significance in the pathways. P value<0.05 and folder change>2 was considered to be criteria for statistically significant and would be selected.5′-TGGCACCACACTTTCTACAATGA-3′ and 5′-AGGGACAACACAGCCTGGAT-3′. Expression levels of target genes were analyzed using the CFX Manager system .Total RNA was extracted from stomach tissues including control, model and CA groups using a TRIZOL reagent  per the manufacturer's instructions. cDNA was synthesized from total RNA (1 µg) using TransScript First-Strand cDNA Synthesis SuperMix kit . Quantitative real time PCR  was performed using a TransStart™ Top Green qPCR SuperMix kit . Primers used to amplify S1Pr1, S1Pr3, SphK1, Got2 and Fabp1 were from Invitrogen  and expression of these transcripts was quantified against the housekeeping gene β-actin, which was amplified using the primers Data are expressed as mean ± SEM. SPSS 19.0 for Windows was used for the statistical analysis. The data were analyzed using the ANOVA, with p<0.05 set as the level of statistical significance.The experimental model of acetic acid injected-induced gastric mucosal damage in rats is often employed to screen the compounds for anti-ulcer activity in that it serves as the leading cause of gastric ulcer in humans The representative total ion chromatograms(TIC) of plasma samples derived from control, model, and CA dose groups in negative modes are presented in http://metlin.scripps.edu/) and compared with the accurate mass charge ratio in some databases, including HMDB (http://www.hmdb.org/), KEGG (http://www.genome.jp/kegg/), LIPID MAPS (http://dev.lipidmaps.org:25424/), and PUBCHEM (http://pubchem.ncbi.nlm.nih.gov/). We can know the probable name of potential biomarkers through the first step. In the present study, 10 potential biomarkers were identified (http://www.chemspider.com/), HMDB and other databases to identify the possible chemical constitutions, and MS/MS data were screened to determine the potential structures of the ions. Sphingosine-1-phosphate (S1P) and stearic acid were taken as examples to illustrate fragments of the structure and the appraisal process. The primary and secondary mass spectrometry information was analyzed by Masslynx  software, compared with database, and ion fragments of 379.2488 (C18H38NO5P) were shown in 7H15NO5P, C11H17O, C4H4NO respectively. Finally, it was speculated as S1P after refering and according to their polarity size. Meanwhile, ion fragments of stearic acid 284.2715 (C18H36O2) (15H32), 143.1359 (C9H19O), 117.0962 (C6H13O2) and 83.0962 (C6H11).The small-molecule metabolites of significant differences  were searched by the software of MPP. The potential markers were identified by using the “ID browser” to search in Metlin database (entified . The pre18H36O2)  were 212The biomarkers described above were proved have close relationship with the formation and treatment of gastric ulcer. The significantly up regulated D-glucose, lysine, Uric acid, pyruvic acid, corticosterone, sphingosine-1-phosphate and the down regulated tryptophan, glycocholate, hexadecanedioic acid, stearic acid were observed in the model group compared with control group . This diTo confirm our metabolomics findings, we need some molecular data, so we identified 5 mRNAs which are related to the 4 potential biomarkers and 2 metabolic pathways with RT-PCR. Sphingolipid metabolism, including S1Pr1, S1Pr3 and SphK1 were examined as showed in More detailed analysis of pathways and networks influenced by gastric ulcer was performed by MPP. The pathways obtained shows in Gastric ulcers in humans recur frequently, and the difficulty in treating them is indicated by the adage “Once an ulcer, always an ulcer” The model of gastric ulcer in rats was successfully reproduced. Plasma samples were analyzed by HPLC/ESI-TOF-MS and multivariate statistical analysis. The results showed that the area of the ulcer and dynamic metabolic profiles after CA treatment were closed to the control group, demonstrating that CA had therapeutic efficacy. According to metabolomics analysis, 10 potential biomarkers and 7 related metabolic pathways were identified in our study. The significantly down regulated D-glucose, lysine, uric acid, pyruvic acid, corticosterone, sphingosine-1-phosphate and the up regulated tryptophan, glycocholate, hexadecanedioic acid, stearic acid were observed in the CA group compared with model group. In addition, folic acid metabolism, fatty acid metabolism,and sphingolipid metabolism and many other metabolism were confirmed to have an impact on gastric ulcer. We have determine the expression of mRNAs related to sphingolipid metabolism and fatty acid metabolism to validate the mechanism. Many other potential proteins, genes, enzymes and bioprocess closed to other pathways need future experiments to validate.By analyzing and verifying the specific early biomarkers of a disease, metabolomics enables us to better understand pathological processes and substance metabolic pathways. We believe that the biomarker and pathway analyses have great potential to explore and clarify the therapeutic action of TCM. In current study, we have characterized biomarker interaction networks involve proteins, genes, enzymes and bioprocess as shown in Stearic acid, glycocholate and hexadecanedioic acid changed cause fatty acid metabolism disorder closing to the incidence and rehabilitation of gastric ulcer Metabolomics is one functional level tool being employed to investigate the complex interactions of metabolites with other metabolites (metabolism) but also the regulatory role metabolites provide through interaction with genes, transcripts and proteins. Potential roles for metabolomics in the clinical trials of gastric ulcer include biomarker discovery and validation, molecular target discovery, therapy decisions. Metabolomics has already shown promise in identifying metabolite based biomarkers in gastric ulcer as biochemical profiling tools to provide important insight into the biology of gastric ulcer The potential application of systems biology in medicine is infinite and will have a significant impact on TCM, clinical research and drug development. Metabolomics represents an emerging and powerful discipline that provides an accurate and dynamic picture of the phenotype of biosystems through the study of potential biomarkers of gastric ulcer that could be used for therapeutic targets and discovery of new drugs. In this study, for the first time, we report a comprehensive analysis of metabolic patterns of the treatment of acid-induced gastric ulcer with CA. The action mechanism of CA was analyzed by an effective approach of metabolite profiling, and we have identified 10 differential metabolites associated with gastric ulcer. More importantly, according to the 10 differential metabolites, 7 related pathways were discovered. Particularly, fatty acid metabolism and sphingolipid metabolism were found as the most altered functional pathways associated with gastric ulcer according to related gene epression analysis. Compared with the alterations of gastric ulcer related metabolites, most of them were reset to a healthier level after CA administration. Our findings also show that CA exhibited preventive efficacy against gastric ulcer by adjusting these multiple metabolic pathways to their normal state and may be mediated through protein, gene, enzyme, and bioprocess. Based on our findings, this makes these pathways possible therapeutic targets for advanced gastric ulcer. In conclusion, the results contribute to a further understanding of gastric ulcer mechanisms. In addition, this study of potential metabolites could be used to achieve multiple targets for treatment of gastric ulcer, that lay foundation for finding therapeutic targets and discovering new multi-target drugs.


Background. High-quality clinical and genetic descriptions are crucial to improve knowledge of orofacial clefts and support specific healthcare polices. The objective of this study is to discuss the potential and perspectives of the Brazilian Database on Orofacial Clefts. Methods. From 2008 to 2010, clinical and familial information on 370 subjects was collected by geneticists in eight different services. Data was centrally processed using an international system for case classification and coding. Results. Cleft lip with cleft palate amounted to 198 (53.5%), cleft palate to 99 (26.8%), and cleft lip to 73 (19.7%) cases. Parental consanguinity was present in 5.7% and familial history of cleft was present in 26.3% subjects. Rate of associated major plus minor defects was 48% and syndromic cases amounted to 25% of the samples. Conclusions. Overall results corroborate the literature. Adopted tools are user friendly and could be incorporated into routine patient care. The BDOC exemplifies a network for clinical and genetic research. The data may be useful to develop and improve personalized treatment, family planning, and healthcare policies. This experience should be of interest for geneticists, laboratory-based researchers, and clinicians entrusted with OC worldwide. Accurate and detailed phenotype description of orofacial clefts (OC) is crucial to produce good etiological and epidemiological studies. In this regard, attention should be given to subphenotypic features of the lip , and palate . Similarly important is the screening of minor and major associated defects which has prevalence rate that ranges from 8% to 75%. Although there are true population differences, methodological factors such as sample source and size, method of ascertainment, case definitions, inclusion criteria, coding system, and case classification account for much of this wide variation –13.In the postgenomic era capturing and processing information on human genetic variation, gene-environment interactions, and genotype-phenotype correlations are essential to develop personalized interventions. This has been reinforced by the Human Variome Project (HVP), an international effort launched in 2006. The HVP aim is to develop and make knowledge housed within linked databases on genes, mutations, and variants accessible to the research and medical communities , 15.Databases may also serve as tools in educating health professionals, policymakers, and the general public towards prevention of unnecessary suffering, improvement of healthcare, and elimination of erroneous beliefs that still remain in some cultures. On humanitarian and ethical grounds, these should be ultimate reasons for birth defects research –19.Recognizing the impact of OC, the World Health Organization assigned the coordination of the International Perinatal Database of Typical Orofacial Clefts (IPDTOC) to the International Clearinghouse for Birth Defects Surveillance and Research (ICBDSR), in 2002 –20.Global Registry and Database on Craniofacial Anomalies [As stated in the report nomalies , the quanomalies .Care for people with OC in Brazil has been funded by the government through the Unified Health System  since 1993. In 1998, the National Health Ministry (NHM) created the Brazilian Reference Network for Craniofacial Treatment (RRTDCF). These measures, however, were not preceded or followed by assessment of specific characteristics and impact of OC on the Brazilian population. Currently the RRTDCF numbers 21 units, but just five of them count with geneticist/dysmorphologist in the team , 21, 22.Brazil is a continental country of more than 180 million inhabitants with diverse genetic background and multicultural profile. There is still a shortfall in epidemiological data on overall birth defects in the country. Similar to other parts of the world, data recorded through birth certificates has been criticized on the grounds of ascertainment, sensitivity, reliability, completeness, and consistence of the reports –25.According to the Latin-American Collaborative Study of Congenital Malformations (ECLAMC), the Brazil's birth prevalence of cleft lip (CL) is 49/10,000, cleft lip with cleft palate (CLP) is 116/10,000, and cleft palate (CP) is 58/10,000 . ECLAMC Besides ECLAMC, some cleft services and hospitals linked or not to the RRTDCF record data according to their research field of interest. Therefore, they may include epidemiological, morbidity, mortality, clinical, genetic, and outcome issues. Information gathered, however, is not standardized .High quality of clinical and genetic descriptions is crucial to improve knowledge on OC and support specific healthcare polices. The development and implementation of the Brazilian Database on Orofacial Clefts (BDOC) reported here is a pioneer nationwide initiative to fill in the gap on clinical and genetic information on OC in the Brazilian population.The aims of this study are to report the implementation, to describe preliminary clinical and familial characteristics and to discuss the potential, and perspectives of the BDOC. BDOC is a nationwide, hospital-based, voluntary and primary database. Initially, a 10-year schedule was planned to run as a continuous and flexible system in which new aims and tools can be aggregated according to the experience gained. General planning of activities started in 2006 and the validation of the tools started in 2008 in voluntary hospitals with clinical geneticists. According to the strategy originally proposed, after this phase, other hospitals without geneticists could be invited to participate. Clinical and laboratory data are updated during the followup of each subject. The database was approved by the local Institutional Review Boards and the National Research Ethics Committee (CONEP # 14733). All subjects provided informed consent.Standardized individual and familial information forms the core database. It is complemented by other five satellite protocols designed to cover the following issues: (1) genetic (Biobank of DNA); (2) morbidity and mortality; (3) services' structure and dynamics; (4) professionals' educating characteristics and protocols; and (5) subject/parent satisfaction. Core database was initiated in eight sites comprising three units of the RRTDCF, two multiprofessional non-RRTDCF centres, and three genetic services. These sites were invited because they all have geneticists with clinical experience in dysmorphology. All of them were personally visited by the coordinators (ILM and VLGSL) before starting the collection of data. Individuals with typical OC and Pierre-Robin sequence in isolated and nonisolated presentation were included. Data on abortuses, stillbirths, cleft uvula, median, oblique, and submucous clefts were not included.isolated and associated were used to refer to additional minor or major defects regardless of the cause or mechanism involved while syndromic and nonsyndromic, to refer to the underlying aetiology [Typical OC  and Pierre-Robin sequence were defined according to the International Classification of Diseases 10th Edition. Terms etiology .http://www.icbdsr.org/. Case classification was based on the definitions of the IPDTOC Working Group (2011) which defines three phenotype categories: isolated clefts, recognized syndrome, and multimalformed cases (MMC). Accordingly, cases of known nonrandom association  are included into the category of recognized syndromes. Cases with random combination of major unrelated defects with evidence of distinct aetiological factors are included in the group of MMC. Deformities were considered minor defects . A list Data were collected using a paper-based record form specifically designed for this database according to the operating manual. These tools were based on the “US National Birth Defects Prevention Study” and ESF “common core protocols” for cleft research and developed as part of a previous study .The record form comprises 80 questions which cover the following information: (1) obstetric, birth, neonatal, and medical history; (2) socioeconomic status; (3) family history where possible ; (3) type of cleft according to topography , severity  and laterality (left and right sided), (4) morphological assessment (including verbatim description of dysmorphic features); (5) laboratory tests  and/or molecular analysis, biochemical tests, ultrasound, X-ray images, and so forth; and (5) diagnosis and its evidences. Operating manual includes the following content: (1) diagram for phenotype categorization, (2) operating definitions, (3) clinical descriptors, (4) list of related defects, (5) list of minor defects, (6) examples of twinning, (7) examples of toxic and occupation-related substances, (8) examples of consanguineous relationships, (9) instructions for taking standard photographs, and (10) examples on how to fill the form.All subjects were personally interviewed and examined by the participant geneticist during routine genetic evaluation from November 2008 to December 2010. As the major proposal of this database is to collect clinical and familial information, there were no restrictions regarding age and existence of previous cleft surgery at the time of enrolment.Forms were sent by post or delivered in person for data reviewing and coding at Unicamp where the BDOC is seeded. The data manager checked all the information received and sought for clarifications when needed. Before entering data into the electronic database, all record forms were manually reviewed and coded by the coordinators. Record form and operating manual were pretested by seven geneticists throughout a six-month period. A total of 143 record forms and 10 assessment questionnaires were included. Mean time spent to complete the record form at the end of this phase was 20 minutes (SD = 4.57) .All geneticists asked for revision of wording, spacing, and ordering of some questions. Coordinator centre asked for further information and clarification of some responses in 28 record forms. Despite this, record form and operating manual were assessed as useful and reliable tools [At the end of this phase a unit of the RRTDCF ceased its participation and, a new site, a genetic service, was included. All participants attended biannual meetings to exchange experience, discuss data, and plan the next stages. t-test, while variables with nonnormal distribution were tested using Kruskal-Wallis and Mann-Whitney tests. The significance level of 5% (P < 0.05) was adopted for all tests.The electronic database was built using Microsoft Access version 2007. Data processing and analysis were performed using two statistical packages (SPSS for Windows version 15.0 and EpiInfo versions 3.5.1 and 3.04d). Categorical variables were analysed using chi-square test. Numerical variables were tested for normality using one-sample Kolmogorov-Smirnov test. Variables with normal distribution were tested using ANOVA and Student's Demographic, clinical, and familial information of 370 individuals with OC was prospective and systematically recorded in the sites identified in Majority of subjects (63.2%) were living in the northeast of Brazil. Males amounted to 219 (59.2%) while females to 151 (40.8%). Birth weight was available in 324 cases. It ranged from 1195 g to 4900 g . Age ranged from 0 to 50 years .P < 0.001) and had lower birth weight (P = 0.047) than those seen elsewhere.Distribution of subjects according to type of cleft  with regard to gender, severity, laterality, and presence of additional defects is shown in P = 0.0001) and of females among those with CP . Bilateral clefts were more frequent for subjects with CLP than for those with CL (P = 0.0002). Chi-squared contingency table revealed significant differences among all groups of clefts. There was an excess of males among individuals with CLP + CL , type of cleft (P = 0.451), and phenotype category  (P = 0.381). Parental consanguinity was present in 21/368 (5.7%) cases, 10 of which were first cousins. Consanguineous marriages were not statistically associated with geographic origin (P = 0.005) but not with CL (P = 0.183).Excluding 12 individuals from whom familial data were not available, familial history of cleft was found in 94/358 (26.3%) cases. First-degree relatives were affected in 18 (19.4%), second degree in 12 (12.9%), and third degree and above in 63 (67.7%) families. There was an excess of affected relatives in the CLP subgroup in comparison with CP (P = 0.193). On the other hand, major defects were more likely to be found in the groups of CLP and CP and unlikely in the group of CL (P = 0.003).Global rate of associated defects (minor plus major) was 179 (48.4%). Fifty-nine (15.9%) subjects showed both minor and major, 103 (27.8%) had only minor and 17 (4.6%) had only major-associated defects. As shown in Ninety-three (25%) subjects were classified as having syndromic clefts. Comparisons between nonsyndromic and syndromic cases are presented in P = 0.006) and statistically more associated with CP (P = 0.000). These cases also showed lower birth weight in both categorical (P = 0.001) and quantitative analyses (P = 0.000) and higher mean of minor defects (P = 0.000). Syndromic cases were more likely to be found at genetic services (minor defects (P = 0.000). Anatomic distribution of minor and major defects is shown in Figures Subjects were regrouped according to the number of minor defects into two categories 1–3 and ≥4 defects). There was a predominance of syndromic cases in the subgroup with four and above  defects.major defects. Based on verbatim description, the following phenotype categories were identified: 277 (75%) isolated clefts, 47 (12%) recognized syndromes or associations, and 46 (13%) multiple malformed cases . Eighteen = 21; 45%), followed by chromosomal , heterogeneous , and teratogenic categories . The 47 clinical recognized syndromes and associations were categorized according to aetiology. Mendelian syndromes were in the lead . The northeast region counts with a site of each category of service . The genetic service from the last-mentioned region was conducting a parallel study on OC which justifies its high amount of subjects . Taking Study design allowed a wide range of subjects' age at enrolment. Younger individuals were preferentially seen in multidisciplinary non-RRTDCF and RRTDCF sites. Subjects enrolled at genetic units were among the oldest, suggesting that the subjects primarily refer to surgery treatment instead of genetic evaluation. In addition, previous studies , 31 haveThe overall results on type of cleft, gender ratio differences, severity  and laterality (left and right sided) of the lip defects corroborate the literature , 32, 33.Prevalence of consanguinity in this sample was higher than that reported for the Brazilian population –36 and dIn the present study, more than one in four subjects showed family history of OC and almost one in five had an affected individual among their first degree relatives. In this subgroup, CLP was the most prevalent followed by CL and CP. A population-based study conducted in Denmark showed that anatomical severity does have an effect on recurrence in first-degree relatives and the type of cleft is predictive of the recurrence type. Third-degree relatives also have an increased recurrence risk compared to the background population .In our sample, global rate of associated defects was around 48% and predominated among subjects with CP. Minor defects were more prevalent in craniofacial region, while cardiovascular and central nervous systems were mainly and almost equally affected by major defects. One out of four individuals was assigned as a syndromic case. Proportion of syndromic cases was higher among individuals with CP and lower among those with CL. Methodological differences regarding case definition, inclusion or exclusion of minor defects, and anomalies/syndromes grouping-system hinder comparisons with many published data. Despite this, our results on global rate of associated defects and anatomic regions involved are similar to those reported by previous studies , 13, 32.IRF6, VAX1, and 8q24 locus have a confirmed role in nonsyndromic OC. Environmental factors, lifestyle, and the preventive role of vitamin supplements have been also investigated. Maternal smoking during pregnancy is consistently linked with increased risk of OC. Findings on the other risk factors and gene-environment interactions, including folic acid, have been inconclusive due to methodological issues [Among more than 20 investigated genes, l issues . Besidesl issues .Despite important advances in the understanding of nonsyndromic OC, around 50% of patients with syndromic pictures remain as cases of multiple congenital anomalies without an identifiable aetiological factor. Laboratory facilities have improved the rate of specific diagnosis so that more than 600 syndromes involving OC have been already recognized. Chromosomal aberrations are the most frequent aetiological group, followed by Mendelian/heterogeneous abnormalities and teratogenic factors , 41, 42.Numeric and structural chromosomal abnormalities, including 22q11 deletion, were detected in 13 cases. Mendelian, heterogeneous, and teratogenic conditions were diagnosed on the basis of clinical evidences. Limited laboratory facilities are challengeable and the Brazilian database may be helpful to define which tests are critical to our population. Collaboration to make these tests available into the network would be economically advantageous. This is an important strategy for healthcare planning , 43–45.As knowledge of genetics and of gene-environment interaction in the aetiology of OC improves, clinical genetics is becoming increasingly important specialty to ensure accurate diagnosis and allow appropriate genetic counselling . TherefoThe process of interpreting clinical data to determine whether an individual has the defect of interest as syndromic or nonsyndromic defect is complex and involves some degree of subjectivity , 46. Metcranface@fcm.unicamp.br); (4) experience gained throughout the process is shared among participants in face-to-face biannual meetings which improve enthusiasm and cohesiveness of the group.The experience reported here shows how a group of geneticists has developed, implemented, and maintained a network suitable for clinical and genetic research on OC. Strengths of this study are that (1) information is prospectively collected by geneticists experienced in dysmorphology following a standard method and strictly defined protocol; (2) data is centrally storage and processed following a defined stepwise approach which uses IPDTOC/ICBDMS definitions, descriptors, and code system; (3) case record form and operating manual are user-friendly tools and may be incorporated in the routine in other cleft centres, these tools are available to interested researchers through contact with the Cranio-Face Brazil Project (Approaches with new technologies such as Genome Wide Association Studies (GWAS) and open array using accurate clinical data probably would bring interesting results to improve knowledge on the aetiology of OC. Besides research applications, information gathered in the BDOC may be useful to develop and improve personalized treatment, family planning, and public health policies on clinical and laboratory genetic investigation. This should be of interest not only for geneticists and laboratory-based scientists but also—and perhaps especially—for policymakers and clinicians entrusted with OC worldwide.


Flawed ABC transporter functions may contribute to increased risk of drug-induced liver injury (DILI). We aimed to analyse the influence of genetic variations in ABC transporters on the risk of DILI development and clinical presentations in a large Spanish DILI cohort.ABCB1 , ABCB4 (1954A>G) and ABCC2  were genotyped using Taqman 5′ allelic discrimination assays or sequencing in 141 Spanish DILI patients and 161 controls. The influence of specific genotypes, alleles and haplotypes on the risk of DILI development and clinical presentations was analysed.A total of ten polymorphisms in ABCC2 −1774del allele were however only found in DILI patients. Hence, this genotype could potentially be associated with increased risk, though its low frequency in our Spanish cohort prevented a final conclusion. Furthermore, carriers homozygous for the ABCC2 −1774G/−1549A/−24T/1249G/3972T/4544G haplotype were found to have a higher propensity for total bilirubin elevations when developing DILI.None of the individual polymorphisms or haplotypes was found to be associated with DILI development. Carriers homozygous for the ABCB1, ABCB4 and ABCC2 transporter genes in DILI development in Spanish patients. The ABCC2 −1774deldel genotype was however restricted to DILI cases and could potentially contribute to enhanced DILI susceptibility.Our findings do not support a role for the analysed polymorphisms in the  Drug-induced liver injury (DILI) is an adverse drug reaction of special concern due to its potentially severe consequences. Despite increased research in this area over the last decades, the underlying mechanisms of DILI are not fully understood. However, host genetic variations are believed to be essential components in DILI development. Many DILI studies to date have focused on variations in genes involved in phase I (bioactivation) and II (detoxification) of drug metabolism and several potential risk alleles have been identified in vitro models has been shown to be highly predictive of liver injury in drug development ABCB11, which encodes BSEP, has been linked to the risk of hepatocellular DILI in a large population of DILI patients from a Spanish Registry Among the transporters the most investigated for its relationship with DILI is the bile salt export pump protein (BSEP), which mediates the efflux o bile acids from hepatocytes into the bile canaliculus. Interference with BSEP function using ABCB1 gene, which contains 29 exons in a genomic region spanning 209.6 kb ABCB1 has been suggested to play a role in specific forms of hepatotoxicity Less is known regarding the role of other transporters in hepatotoxicity. P-glycoprotein (P-gp) is the major drug transporter recognizing a wide range of substrates. In the liver P-gp is located in the canalicular membrane, where it facilitates biliary drug excretion. This transporter is encoded by the ABCC2 gene, is likewise involved in drug transport. It is confined to the apical membrane of various polarized cells and subsequently involved in the final stage of cellular detoxification. Similar to P-gp, this ABC transporter has broad substrate specificity, comprising of many conjugated organic anions and drug metabolites. In addition, MRP2 also plays an essential part in bilirubin glucuronoside and bile salt sulfate/glucuronoside biliary excretion ABCC2 polymorphisms in DILI is currently unclear with controversial results between studies The multidrug resistance protein 2 (MRP2), encoded by the ABCB4 gene and highly expressed in the liver. This ABC transporter is not believed to play a major role in drug transport, but is essential for biliary phosphatidylcholine (PC) secretion. PC combines with bile salts to form mixed micelles, which prevent against damage caused by detergent-like bile salts activity. With insufficient PC concentration, bile salts transported into the bile by BSEP can potentially solubilize cell membranes resulting in biliary toxicity Multidrug resistance protein 3 (MDR3) is encoded by the ABCB1, ABCB4 and ABCC2. The ABCC2 polymorphisms were selected based on previous DILI association studies in ethnically different cohorts ABCB1 polymorphisms may affect serum drug concentration ABCB4 polymorphism changes an evolutionary conserved amino acid, which could lead to altered protein physiochemical properties Due to the participation of ABC transporters in drug elimination and their known capacity to produce serious hepatic conditions A total of 141 DILI cases were selected from those submitted to the Spanish DILI Registry, a collaborative network established in 1994 to prospectively identify cases of DILI in a standardized manner. The criteria for DILI at the time of subject inclusions in the study were: an increase in alanine transaminase (ALT) ≥3 times the upper limit of normal (xULN) or ≥2 xULN of alkaline phosphatase (ALP). However, 93% of the cases also fulfilled the more recent DILI criteria established by Aithal and coworkers: ALT ≥5 xULN or ALT ≥3 xULN + total bilirubin (TBL) ≥2 xULN or ALP ≥2 xULN The study protocol was approved by the local ethics committee of the coordinating center at the Hospital Universitario Virgen de la Victoria (Comité Ético de Investigación) in Málaga, Spain. All subjects who took part in the study were over 18 years of age. All subjects gave written informed consent, which conformed to the current Helsinki Declaration, prior to study enrolment.ABCB1 c.1236T>C , c.3435T>C , ABCB4 c.1954A>G  and ABCC2 c.-1549A>G (rs1885301), c.-24C>T (rs717620), c.1249G>A , c.3972C>T  and c.4544G>A  using a validated 5′-nuclease PCR based assay with allele specific fluorescent probes  as previously described Venous blood was obtained from each subject and DNA was extracted as described previously ABCB1 2677G>T/A  and ABCC2 -1774G>del (rs369192412) polymorphisms, these were genotyped by direct sequencing with Big dye terminator cycle sequencing kit  of a 224 and 554 basepair amplicon, respectively, encompassing the polymorphism of interest. The primers used for sequencing were 5′-AGGTTCCAGGCTTGTTGTAATTAC-3′/5′-GGAAGAACAGTGTGAAGACAATGG-3′ (rs2032582) and 5′-GAAAATGATGGAGAAACTGCCATA-3′/5′-ATGACAACTGAATCAACTTCACAA-3′ (rs369192412).Due to the nature of the P) was multiplied by the number of genotypes or alleles compared to give a corrected P value (Pc), was performed in order to account for problems of significant associations arising by chance after multiple comparisons. Haplotype frequencies were performed for the ABCC2 and ABCB1 polymorphisms using the Haploview 4.1 software . Due to software incompatibility with three allelic polymorphisms the ABCB1 2677G>T,A was considered as 2677G>T. Hence, six cases and seven controls with an ABCB1 2677A allele were omitted from the haplotype analysis. Allele combination frequencies formed by ABCC2/ABCB1 and ABCB1/ABCB4 in 97 DILI patients and 125 controls were also predicted using Haploview 4.1.Genotype distributions and allelic frequencies were analysed in DILI patients and controls using the comparison of proportions test, a derivative of the Fisher's exact test. Bonferroni's correction for multiple tests, whereby the probability value .Means were compared by Student's t test for independent sample. Analysis of variance (ANOVA) was used for comparison of groups. Where variables did not follow a normal distribution, a nonparametric Kruskal-Wallis analysis was performed. All analyses were performed using the SPSS 19.0 statistical software package  and A total of 141 DILI patients were included in the study of which 73 were males and 68 females. The mean age at DILI onset was 54 years, ranging from 14 to 83 years. The predominant pattern of injury, based on the R value calculated from the first blood sample analysis after DILI onset, was hepatocellular (n = 77) followed by mixed (n = 36) and cholestatic (n = 28). The main causative therapeutic drug group was antiinfectives (37%), followed by musculo-skeletal (13%), nervous system (12%), cardiovascular (11%) and antineoplastic drugs (7%). Of the 141 patients only one developed fulminant hepatic failure and required a liver transplantation. Applying the Council for International Organizations of Medical Science (CIOMS) causality scale the 141 cases were classified as 78 (55%) highly probable, 56 (40%) probable and 7 (5%) possible DILI cases. Demographics, clinical and laboratory parameters of the 141 DILI cases included in the study are outlined in ABCB1 , ABCB4 (1954A>G) and ABCC2 . No significant differences were found in either genotype distribution or allele frequency between cases and controls for any of the individual polymorphisms (ABCB1 2677G>A (24%), ABCB4 1954A>G (65%) and ABCC2 -1774G>del (65%) and 4544G>A (59%). The ABCC2 -1774deldel genotype was not present in any of the controls, which prevented a statistical analysis using the comparison of proportions method. However, when analysing the ABCC2 -1774deldel distribution between cases and controls using the Fisher's exact test the difference was found to be statistically significant (p = 0.014). As the MRP2 transporter, encoded by ABCC2, is known to transport various conjugated drug metabolites a smaller subgroup of 22 DILI cases induced by drugs undergoing glucuronidation was also analysed. None of the alleles corresponding to the six ABCC2 polymorphisms were however found to correlate with DILI risk in this subgroup (data not shown).In search for genetic differences DILI patients and controls were genotyped for 10 specific polymorphisms located in orphisms . The staABCC2 polymorphisms in particular among -1549A>G, -24C>T and 1249G>A, which formed a haplotype block  were found to have a higher tendency towards elevated TBL values with a mean value of 12.2 xULN vs 6.5  and 6.1  (p = 0.06) at presentation. The -24TT carriers also demonstrated higher maximum TBL, although not reaching statistical significance (TT: 12.5 vs CC: 7.2 and CT: 7.1 xULN) (p = 0.23). Seventy percent of the TT carriers reached a maximum TBL value of more than 10 xULN, while the corresponding proportion for the CC and CT carriers were 25% and 21%, respectively (p = 0.011) (p = 0.0074) as well as CT and TT carriers (p = 0.0059) in terms of proportion of cases with bilirubin levels above 10 xULN. In addition to being more prone to higher TBL elevations, all the ABCC2 -24TT carriers shared the same ABCC2 diplotype, being homozygous for the -1774G, -1549A, -24T, 1249G, 3972T and 4544G alleles and 73% developed hepatocellular type of injury.Phenotypic parameters, such as type of liver injury, serum total bilirubin (TBL), alanine transaminase (ALT) and alkaline phosphatase (ALP) values, at presentation were analysed according to genotypes in the 10 different polymorphisms. Patients with the = 0.011) . Post hoABCB1, ABCB4 and ABCC2 polymorphisms.Patients homozygous for the 1249A allele (n = 7) were all found to have developed hepatocellular injury, but only 29% reached a maximum TBL value of more than 5 xULN. All of these patients were also homozygous for the -1549G and -24C alleles with the majority being homozygous for the -1774G, 3972C and 4544G alleles. No further clinical or biochemical parameters were found to differ between genotypes and alleles for any of the analysed ABCB4 and ABCC2, respectively. In this study we analysed a total of 10 polymorphisms located in ABCB1, ABCB4 and ABCC2 in DILI patients and healthy controls and found that none of the individual polymorphisms were associated with DILI development. The statistical power to detect differences with an odds ratio of 2 was relatively high (>75%) for most of the polymorphisms. However, in the polymorphisms with lower minor allele frequencies and consequently lower statistical power, in particular ABCB1 2677G>A and ABCC2 4544G>A, it is possible that this may have contributed to the inability to detect significant differences between the cases and controls. Hence, to ascertain that these polymorphisms have no role in DILI development, despite our findings, requires larger cohorts. Furthermore, our controls were not drug-matched and although healthy at the time of the blood extraction (and having no recorded DILI episode) the possibility that some of them could develop DILI at a later stage cannot be absolutely discarded. Due to the low incidence rate of idiosyncratic DILI (1/10 000–1/100 000 user of a specific treatment) however, we hold it for very unlikely that a large proportion of the controls would be susceptible to developing DILI. In addition, the main causative agents in our DILI cases were antiinfectives and musculo-skeletal drugs, which are widely used in the Spanish community. Hence, it is likely that many of the controls in fact have been exposed to the causative agents corresponding to the DILI cases. In an earlier study we compared genotype data of an additional ABC transporter polymorphism in this control cohort with those of controls with recorded drug intakes and found no significant differences between the two cohorts Based on their crucial role in drug metabolism and maintenance of liver homeostasis ABC transporters could play a role in DILI development. A defect in any of these transporters can affect biliary clearance and subsequently delay the elimination of toxic substances, including drug metabolites. This has been confirmed through diseases such as intrahepatic cholestasis and Dubin-Johnson syndrome caused by mutations in ABCC2 -1774del carriers as this genotype was not present in any of the controls. Evidently, this needs to be confirmed in a larger cohort, which could be a difficult task due to the rarity of this genotype in conjunction with DILI itself being a relatively rare condition. Nevertheless, the -1774deldel genotype has been found to play a role in cholestatic/mixed type of DILI development in a Korean population, where this genotype is more common than seen in Caucasians We found, however, that there was a tendency towards a DILI association for homozygous ABCC2 -1549A and 3972T alleles appear to be more prone to developing hepatocellular type of DILI ABCC2 risk alleles were identified, contained mostly herbal remedy-induced hepatotoxicity cases, while our cohort included a larger variety of causal agents, mainly conventional pharmaceuticals. The presence of drug-specific risk alleles is supported by a recent analysis of seven ABCC2 polymorphisms in a different Korean cohort consisting of antituberculosis treatment-induced hepatotoxicity cases. This study could not reproduce the results found in the earlier Korean cohort, as no associations were found for any of the individual polymorphisms or haplotypes ABCC2 -24C>T genotype distribution between British DILI patients and controls Koreans homozygous for the ABCC2 haplotypes were found to be over- or underrepresented in the DILI cohort. However, a smaller group of patients with substantial TBL elevations were identified based on sharing an identical diplotype (-1774GG/-1549AA/-24TT/1249GG/3972TT/4581GG). The ABCC2 -24T/1249G/3972T haplotype has been shown to yield lower protein expression and efflux rate in vitro compared to the more common -24C/1249G/3972C haplotype, presumably due to variations in mRNA secondary structures and resultant posttranscriptional modifications ABCC2 haplotype -1774G/-1549A/-24T/1249G/3972T/4544G has a similar functional effect, based on indications of reduced expression for the -1549A and 4544G allele ABCC3, and consequently elevating serum concentrations of bilirubin glucuronosides. Conjugated bilirubin transport by MRP3 has been demonstrated and hepatic MPR3 induction has been found in situations of decreased MRP2 activity No ABCB1 polymorphisms focused on in this study are known to vary considerably between different ethnic populations. However, our results were comparable to those established earlier in Caucasian populations ABCB1 polymorphisms and DILI development, though the ABCB1 3435T allele has been associated with reduced risk of nevirapine hepatotoxicity in several independent studies Allele distributions of the ABCB1 polymorphisms studied were found to be in linkage disequilibrium with low haplotype diversity. The two major haplotypes 1236C/2677G/3435C and 1236T/2677T/3435T were predicted to be present in approximately 80% of the cohort. The difference between ABCB1 haplotypes in terms of transporter activity is unclear. Our study, however, does not support evidence for ABCB1 haplotypes having differential effects on DILI development. Interestingly the ABCB4 1954A>G and ABCB1 3435T>C SNP pair were seen to have a high D' value, suggesting possible LD, though this is not supported by the low R2 value. Due to the low frequency of the ABCB4 G allele and the large distance between the SNPs (>80 kbp) this finding should be considered with caution. Nevertheless, long distance LD has been reported in ABCB1 stretching across to the nearby ABCB4 gene In accordance with previous results the three ABCB1/ABCC2 allele combinations effecting DILI development may require DILI cases with the same causative agent or those forming similar metabolites in terms of structural elements. Hence, the pooling of DILI cases induced by a wide range of causal agents could have diluted potentially different allele combination frequencies.Both MRP2 and P-gp have wide substrate specificity with certain degree of overlap, in addition to their co-localization on the hepatocyte canalicular membrane. We therefore hypothesised that reduced activity in one of these transporters may be compensated for by the other. Hence, specific allele combinations formed by these two genes could potentially shed light onto the underlying mechanism of DILI development. It is possible that putative ABCB1, ABCB4 and ABCC2 in DILI development in Spanish patients, neither individually nor in combinations. The -1774del allele in ABCC2 was however restricted to DILI cases and could potentially contribute to enhanced DILI susceptibility. In addition, this study highlights the differential effect of specific ABC transporter alleles in ethnically different cohorts.In conclusion, due to the complexity of the human efflux transporter system it is difficult to determine its potential role in DILI development. Our data do not support a role for the selected polymorphisms in ABC transporter genes


Primary Biliary Cirrhosis is known to be associated with Urinary Tract Infections (UTIs), but whether these precede or follow the liver disease is unclear. We have therefore attempted to determine whether UTIs are more common in people with Primary Biliary Cirrhosis (PBC) prior to their diagnosis.We conducted a case control study in the General Practice Research Database. All cases of PBC first recorded at least one year after entry to the dataset were selected along with up to 10 controls matched for age, sex. A second unmatched control group who had Chronic Liver Diseases but not PBC were chosen. The main exposures studied were the occurrence of Urinary tract infections and pyelonephritis at least one or at least five years before diagnosis. We also performed an analysis restricted to those younger than 55 at diagnosis, as we hypothesized the relationship to be stronger in the younger age group.PBC is associated with UTI prior to diagnosis, OR 1.50 (CI 1.26-1.78), which was similar 5 years prior to diagnosis and after adjusting for smoking. The strongest relationships were observed in pyelonephritis exposures five years before diagnosis in cases under 55 years: adjusted odds ratios were 2.60 (1.02-6.63) in comparison with matched general population controls and adjusted odds ratios were OR 2.45 (1.02-5.59) in the comparison with chronic liver disease controls.We found that the association between urosepsis and PBC is specific to this disease and precedes the diagnosis of PBC in a manner not previously observed in human data. This is consistent with a causal relationship. Primary Biliary Cirrhosis (PBC) is an idiopathic chronic liver disease associated with destruction of small intrahepatic bile ducts . Both geNovosphingobium.aromaticivorans[Escherichia .Coli[There has been recent epidemiological interest in the spatio-temporal clustering of cases, which might indicate involvement of a transient toxic or transmissible agent ,5. The aicivorans. Anothericivorans. A numbehia .Coli,11 whichhia .Coli, and it hia .Coli. Epidemihia .Coli and fourhia .Coli-18. Theyhia .Coli, such spWe have therefore set out to establish whether the epidemiology of the relationship between PBC and UTI is consistent with a causal relationship both by better defining the temporal relationship between PBC and UTI and by establishing whether any relationship is specific to PBC among liver disease. To achieve this we have carried out a large population based case-control study nested in the United Kingdom General Practice Research Database.The United Kingdom General Practice Research Database (GPRD) is a large longitudinal database of primary care records with data from 1987 until the present .We constructed an individually matched case control study nested within GPRD, and using the same cases a second unmatched case control study. Cases were all incident cases of PBC in GPRD. These were defined as those with READ/OXMIS codes for PBC (READ Primary Biliary Cirrhosis or OXMIS code PRIMARY BILIARY CIRRHOSIS (LIVER)) or Anti-mitochondrial antibody (AMA) alongside another code for chronic liver disease or cholestatic liver function tests with AMA or PBC first recorded at least one year after the start of the subject's up to standard data from January 1987 to October 2008. General population controls matched for age, sex and practice were selected at a ratio of approximately 10:1. A second group of unmatched controls who had Chronic Liver Diseases (CLD) but not PBC were selected to permit assessment as to whether findings were specific to PBC or were characteristic of all chronic liver diseases.The primary exposure of interest was the occurrence of urinary tract infection prior to diagnosis of PBC. To study this, we abstracted data on the occurrence of episodes of Urinary Tract Infections (UTI) and of Pyelonephritis from the GPRD record using READ and OXMIS codes for diagnoses. These events were used to categorise subjects as having ever had a UTI or Pyelonephritis occurring at least a year before diagnosis, and occurring at least five years before diagnosis.We considered age, gender, smoking status, and diabetes as potential confounders. For the general population control group, the data was matched for age and gender which are both known to be important risk factors in PBC . For theEscherichia .Coli was lower due to the potential for misdiagnosis of dysuria due to reduced oestrogen levels in this group. Hence we expected to find a weaker relationship in those over 55.All analyses were conducted using STATA10.1/SE. We conducted initial descriptive analyses of the data to produce summary measures of the primary and potentially confounding exposures. Univariate analyses using both general population and CLD controls were conducted using conditional logistic regression for the matched control group and logistic regression for the unmatched controls. We then went on to conduct multivariate regression analyses to assess the effect of smoking in the matched controls. As the CLD controls were unmatched, we also adjusted for age and gender to remove confounding by them. Age and gender were retained in the final model on an a priori basis, but other confounders only if their addition or removal altered the odds ratio by at least 10%. Finally a further stratified analysis was performed by dividing patients into those older than or aged 55 years at diagnosis, and those who were younger than 55 at diagnosis to examine any possible interaction with age. We hypothesised that in post menopausal women, the likelihood that a recorded UTI would accurately reflect infection with The data used to conduct this study was provided via an MRC license. Dr Varyani is an Academic Clinical Fellow funded by the UK NIHR, Dr Card is funded via a HEFCE Senior Lectureship Award and Dr West is an NIHR clinician scientist. None of these funders had any role in the design, conduct, interpretation or writing up of this study.The GPRD Group has ethical approval from a Multi-centre Research Ethics Committee covering all purely observational research using GPRD data (including the current study). The protocol for this project was approved by the Independent Scientific Advisory Committee of the GPRD (protocol number 08_068).We had in total 800 subjects with PBC, 7991 general population matched controls and 12137 unmatched chronic liver disease controls. Overall 87% of PBC cases were female and mean age at diagnosis was 63 years. Cases had a mean follow up time of 6.5 years and both sets of controls had a similar amount of follow up time. The Chronic Liver Disease cohort was younger with a mean age of 51 years and 40% were female. 44% of the PBC patients had ever smoked compared to 37% of the general population controls and 56% of the chronic liver disease control group. 12% of the PBC patients had ever had a diagnosis of diabetes compared with 9% of the controls and 21% of the CLD controls  of PBC patients had a UTI recorded in the dataset at least one year before diagnosis, compared to 1737 (22%) of the general population controls , although a trend was observable in the other exposures assessed. We therefore conducted further analyses stratified by age. Of those patients diagnosed with PBC before the age of 55, 68 31%) had a prior diagnosis of UTI (at least one year prior to diagnosis), compared to 433 (20%) of age and sex matched controls. This corresponds to PBC patients in this age group having odds of prior diagnosis of UTI 1.98 fold higher than controls (CI 1.43-2.75). This figure was virtually unchanged by adjustment of any confounding by smoking, but fell to an OR of 1.36 (1.11-1.66) among those aged 55 or over at diagnosis. When comparing to chronic liver disease controls there was clear confounding by age and gender, and after adjustment for this, the relationships were weaker and ceased to be significant  for matched and 1.61 (1.15-2.25) for chronic liver disease controls). Adjusted ORs for the relationship with Pyelonephritis in those under 55 were 2.60 (1.02-6.63) for matched and 2.45 (1.02-5.59) for chronic liver disease controls , and therefore in essence to the population of the UK. As it is based upon prospectively recorded data, there is no potential for recall bias and since all potential cases for the study were selected, and controls were chosen in an unbiased manner, selection bias also cannot realistically occur.E. Coli infection since this is the commonest UK isolate accounting for about 80% of cases [The great weakness of this as of any study based on an anonymised clinical records database is the potential for error in the definition of both outcomes and exposures which cannot be validated. For the current outcome of PBC, we do not have great concern as to error as we have previously shown that the epidemiology of PBC when described in this database closely mirrors the best modern conventional epidemiological studies available in the UK population ,23-26. Tof cases  , aIn conclusion, we have demonstrated a strong association between UTI and subsequent diagnosis of PBC, that this association goes back at least five years before diagnosis and that it is stronger in younger subjects. In addition, this finding was specific to PBC when compared to other chronic liver diseases. This work provides the strongest evidence yet produced that this relationship is indeed a causal one.AMA: Anti-mitochondrial antibody; CI: 95% Confidence interval; CLD: Chronic Liver Diseases; GPRD: General Practice Research Database; OR: Odds Ratio; PBC: Primary Biliary Cirrhosis; PDC-E2: Pyruvate dehydrogenase complex E2 subunit; UTI: Urinary Tract Infection.The authors declare that they have no competing interests.FKV under supervision by TRC and JW wrote the application for the data, managed the data, undertook the analysis, wrote the first draft, and contributed to the third and all subsequent drafts. JW contributed to supervision of FKV, data management, analysis and interpretation, and contributed to the writing of the third and all subsequent drafts. TRC conceived the project, contributed to supervision of FKV, data management, analysis and interpretation, wrote the second draft and contributed to the writing of the third and all subsequent drafts.All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/11/95/prepub


MCT9/SLC16A9) gene. Although the function of MCT9 remains unclear, urate is mostly excreted via intestine and kidney where MCT9 expression is observed. In this study, we investigated the relationship between a variant of MCT9 and gout in 545 patients and 1,115 healthy volunteers. A missense variant of MCT9 (K258T), rs2242206, significantly increased the risk of ROL gout (p = 0.012), with odds ratio (OR) of 1.28, although it revealed no significant association with all gout cases (p = 0.10), non-ROL gout cases (p = 0.83), and RUE gout cases (p = 0.34). In any case groups and the control group, minor allele frequencies of rs2242206 were >0.40. Therefore, rs2242206 is a common missense variant and is revealed to have an association with ROL gout, indicating that rs2242206 relates to decreased intestinal urate excretion rather than decreased renal urate excretion. Our study provides clues to better understand the pathophysiology of gout as well as the physiological roles of MCT9.Gout is a common disease caused by hyperuricemia, which shows elevated serum uric acid (SUA) levels. From a viewpoint of urate handling in humans, gout patients can be divided into those with renal overload (ROL) gout with intestinal urate underexcretion, and those with renal underexcretion (RUE) gout. Recent genome-wide association studies (GWAS) revealed an association between SUA and a variant in human monocarboxylate transporter 9 ( MCT9/SLC16A9) has a relationship with SUA variation. Although the function of MCT9 is not known, it is a member of the solute-carrier (SLC) transporter that is expressed in several urate-excreting organs, including intestine and kidney. In this study, we investigated the effects of an MCT9 variant on the susceptibility to gout in patients and healthy volunteers.Gout, known for its painful arthritis, is a common disease and a consequence of hyperuricemia , which s2, respectively. Gout patients with high (>25 mg/h/1.73 m2) urinary urate excretion (UUE) MCT9/SLC16A9, gene was performed by TaqMan Assay-By-Design method (Applied Biosystems) with a LightCycler 480 (Roche Diagnostics) [ χ2 test was used for association analysis.Genomic DNA was extracted from whole peripheral blood cells . Genotypnostics) , 14. To nostics) . For allp value for Hardy–Weinberg equilibrium was 0.79, which suggested mistyping was not obtained. As shown in Table p = 0.012), with the odds ratio (OR) of 1.28, although it revealed no significant association with all gout cases (p = 0.10), non-ROL gout cases (p = 0.83), and RUE gout cases (p = 0.34).Table MCT9 gene, is associated with ROL gout but not with overall gout susceptibility. As rs2242206 (in exon 5 of MCT9) is close to rs12356193 (in intron 5), rs2242206 is sometimes used as a substitute for rs12356193, in which a genome-wide association with SUA was demonstrated [MCT9 (K258T), which is a possible dysfunctional mutation. We therefore investigated the relationship between MCT9 and Japanese gout cases using rs2242206.This study shows that rs2242206, a common missense variant in nstrated , 17. In MCT9 rs2242206 and SUA. Although our results show that rs2242206 has no association with overall gout susceptibility, rs2242206 significantly increased the risk of ROL gout. In humans, one third of urate is excreted from the intestine and most of the rest via the kidney. We previously reported that ROL is caused by decreased intestinal urate excretion due to urate transporter ABCG2 dysfunction [MCT9, SUA levels, and metabolites (DL-carnitine and propionyl-l-carnitine), implying that MCT9 indirectly affects extra-renal urate excretion, for instance, by transporting carnitine-related compounds. Whereas further genetic and functional study of human MCT9 is necessary, our study suggests that it has a possible physiological role in urate excretion from human intestinal epithelial cells.A previous study of a small sample (92 participants) by Polašek et al.  revealedfunction  reported