Tumour necrosis factor-alpha (TNF-α) and nuclear factor of kappa light chain gene enhancer in activated B cells (NF-κB) play critical role in carcinogenesis processes like tumour initiation, proliferation, migration and invasion. Single nucleotide polymorphisms in TNF-α, NF-κB and its inhibitor IκB genes were shown to be associated with susceptibility and prognosis of several cancers; however, their role in esophageal squamous cell carcinoma (ESCC) is not well recognised. Therefore, in present study, we aimed to investigate association of common polymorphisms in TNFA, NFkB1 and NFKBIA with risk and prognosis of ESCC in northern Indian population. -308G>A, NFkB1 -94ATTG ins/del and NFKBIA (-826C>T and 3’UTRA>G) polymorphisms using PCR alone or followed by RFLP and TaqMan assay.We genotyped 290 ESCC patients (including 162 followed up cases) and 311 mean age, gender and ethnicity matched controls for TNFA TNFA-308GA genotype was associated with increased risk of ESCC specifically in females and in patients with regional lymph node involvement, while, NFKBIA -826CT+TT genotype conferred decreased risk of ESCC in females. Haplotypes of NFKBIA -826C>T and 3’UTRA>G polymorphisms, C-826G3’UTR and T-826A3’UTR, were associated with reduced risk of ESCC. No independent role of NFkB1 -94ATTG ins/del polymorphism in susceptibility of ESCC was found. Multi-dimensionality reduction analysis showed three factor model TNFA-308, NFKBIA-826, NFKBIA 3’UTR as better predictor for risk of ESCC. Furthermore, combined risk genotype analysis of all studied polymorphisms showed increased risk of ESCC in patients with 1-3 risk genotype compared to ‘0’ risk genotype. Survival analysis did not show any significant prognostic effect of studied polymorphisms. However, in stepwise multivariate analysis, metastasis was found to be independent prognostic predictor of ESCC patients.TNFA-308 and NFKBIA (-826C>T and 3’UTRA>G) polymorphisms may play role in susceptibility but not in prognosis of ESCC patients in northern Indian population.  Chronic inflammation, a critical component of tumour microenvironment, is involved in pathogenesis of approximately 25% of all human cancers including esophageal cancer (EC) ,2. TumouTNFA) spans about 3 kilo-base pair on chromosome 6p21.3 and contains 4 exons. It encodes a pro-inflammatory pleiotropic cytokine that plays role in cell differentiation, proliferation, apoptosis and immunity [TNFA; however the most documented single nucleotide polymorphism (SNP) is located at 308 nucleotide position resulting in substitution of G to A (rs1800629). Epidemiological studies exploring association of TNFA-308 G>A polymorphism with cancer risk are inconsistent. While, some studies have shown increased risk of carcinoma of breast [TNFA-308 polymorphism may also have prognostic implication as it was found to confer adverse outcome to head & neck cancer and gastro-esophageal patients [TNF-α gene , and p52/p100. The dimeric form of NF-κB, p50/RelA, is the most common form [NFKBIA, located on 14q13) is classical form that can be found in the cytoplasm and nuclei [NFKBIA gene in KYSE series EC cell lines [NFKB1 (1900 SNPs) and NFKBIA (158 SNPs) according to dbSNP database (www.ncbi.nlm.nih.gov/snp), however, previous studies have extensively explored role of common polymorphic variants in promoter region of NFKB1  and NFKBIA  and 3’UTR region of NFKBIA  in susceptibility and prognosis of various cancers [NFKB1 and NFKBIA variants in EC are missing till now. So, in the present study, we investigated the association of TNFA-308 G>A, NFKB1 -94ATTG ins/del and NFKBIA (-826 C>T and 3’UTR A>G) polymorphisms with susceptibility to esophageal squamous cell carcinoma (ESCC) or its clinical phenotypes, their interaction with environmental risk factors and their role in survival outcome of ESCC patients.NF-κB is a family of transcription factors that are activated by TNF-α in classical canonical pathway . There amon form . In un-smon form . The IκBd nuclei . NF-κB wd nuclei ,22. Whil cancers . Literatth Sanjay Gandhi Post Graduate Institute of Medical Sciences Ethics Committee Meeting on May 14, 2003) and written informed consent was obtained from all recruited individuals. All the clinical and demographical details were recorded anonymously by one individual (data collector), while experiments and analysis was performed by another individual (investigator). The study subjects were coded in random numbers and identity of patients  was never revealed to investigator at any stage of the study. Blood samples and other details from all study subjects were collected after taking their informed written consent.The study was approved by the ethical committee of Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow  and adenocarcinoma (ADC) were recruited in the study. However, the frequency of ADC cases (6.8%) was low in our study and etiologies of SCC and ADC are entirely different; therefore, we carried our analysis in 290 incident ESCC cases only. Patients with pre-malignant conditions like corrosive esophageal injury, Achalasia injury, Barrett’s esophagus or Gastroesophageal reflux diseass and prior treatment before enrolment were excluded from the study. Data on demography, clinical parameters  and environmental exposures  were recorded only for the patients in a standard proforma from medical records and personal interview as described previously . During For survival analysis, patients who had received radiotherapy (RT) with or without concurrent cisplatin based chemotherapy (CT) were followed up during 2005-2012. Many surgically resected cases and patients who discontinued their treatment could not be followed up, so prognostic evaluation was limited to 162 cases. The inclusion criteria of patients for survival analysis was patients who had received RT based treatment regime i.e. patients who had received RT or RT along with CT. However, patients who were referred for surgery or for palliative treatment or with previous history of surgical resection were excluded in the survival analysis. The maximum follow up time was 72.6 months and the median follow up time was 9.3 months. Dosage of RT and CT, dysphagia grade and duration, tumour stage, Karnofsky performance score and other parameters were recorded for each followed patients. The patients were prospectively followed up every six months from the time of enrolment through telephonic conversations or their visit to out-patient clinic until death or end of the study. The regional lymph node involvement was recorded as ‘present’ or ‘absent’. Around, 47% (120/255) of cases had regional lymph node involvement in overall-group of patients, while, in followed up patients group, regional lymph node positivity was recorded in only 39.5% (64/162) of cases.TNFA-308 G>A and NFKB1 -94ins/del ATTG polymorphisms were carried out through PCR based methods using primer sequences as described previously [UTR A>G polymorphisms were genotyped by TaqMan assay and PCR RFLP [A 3 ml venous blood was collected from each subjects in sterile EDTA vacutainer tubes and stored at -40°C until DNA extraction. Genomic DNA was extracted from peripheral blood leukocytes using standard salting out method . The geneviously ,33. NFKBPCR RFLP  respecti2 test. Binary logistic regression was applied to calculate Odds ratio (OR) and 95% Class interval (CI) for various predictors after adjustment of covariates like age and gender. Haplotype analysis of NFKBIA polymorphisms was conducted using SNPAnalyzer version 1.0 [www.multifactordimensionalityreduction.org). MDR software gives number of output parameters like cross validation consistency (CVC), testing accuracy (TA), balanced training accuracy for different interactions and single best model is identified as interaction that had maximum CVC and TA. Statistical significance of the model was evaluated using a 1000-fold permutation test. Kaplan Meier and Log rank tests were carried out to estimate the difference in survival times according to genotypes and clinical/demographical characteristics. Survival time was calculated from date of ESCC diagnosis to death of patients or date of last follow up. Univariate Cox regression analysis was done to determine predictive factor of ESCC survival by estimating Hazard ratio (HRs) and 95% CI. Multivariate analysis was also performed, in which all variables were first entered together in single step and after that in stepwise manner also, to identify independent prognostic predictor of ESCC. Two models (forward selection and backward elimination) were employed in stepwise Cox regression analysis. All statistical analyses were performed with SPSS software version 15.0  and differences were taken as significant when two sided P-value was less than 0.05.The effective sample size for case-control study was calculated using Quanto 1.1 ver. software , while msion 1.0 . Since rTNFA-308 G>A polymorphism) and genetic effect of 2.0, the case control pair of 240 was sufficient to achieve 80% power. The power calculation analysis showed that at minimum minor allelic frequency (MAF) of 5.4%  of patients had received RT only, while, 70.4% (114/162) of cases had received both CT and RT. The ratio of alive, dead and lost to follow up cases was 25 (15.4%): 76 (46.9%): 61 (37.7%). Most of the patients (71.9%) had tumour either in T1 or T2 stage. Median dysphagia duration was 3 month. The frequency of dysphagia grade 1+2, 3 and 4 was 99 (61.5%), 49 (30.4%) and 13 (8.1%) respectively. Also, majority of patients were tobacco users (77.8%), 48.7% of cases were smokers, 29.9% were alcohol drinkers and 23.5% of patients had occupational exposure in the followed up group. TNFA, NFKB1 and NFKBIA were in accordance with HWE in controls (P>0.05 in each case). When genotypic distribution of TNFA -308 G>A polymorphism was compared between cases and controls, 1.7 fold increased risk of ESCC was observed with TNFA -308 GA genotype compared to GG genotype . Also, TNFA -308 A allele was associated with enhanced risk of ESCC compared to G allele   polymorphisms were observed with the overall risk of ESCC. Chi square test showed genotypic frequencies of polymorphisms in = 0.019) . No assoNFKB1 -94ATTG ins/del, NFKBIA (-826 C>T and 3’UTR A>G) polymorphisms on the survival outcome of ESCC patients   even after FDR test  . However= 0.057) . No maleNFKB1 -94ATTG ins/del and NFKBIA polymorphisms with clinical characteristics  of ESCCAssociation of the TNFA-308 G>A, TNFA -308 or NFKB1 -94 ins/del polymorphisms did not modulate any site-specific risk of ESCC tumours. We also analysed the role of the genetic variants with regional lymph node involvement and found ESSC patients with TNFA -308 GA or GA+AA genotypes were at two fold increased risk of regional lymph node involvement   . TNFA -3= 0.008) . A case only analysis was performed to evaluate interaction of the polymorphisms with environmental risk factors, however, no significant modulation in risk of ESCC was found in tobacco users, smokers and alcohol users (Data not shown). UTR A>G polymorphisms were in moderate LD in controls as well as in patients . A total of four haplotypes were observed in the subjects. The frequency of C-826G3’UTR and T-826A3’UTR haplotypes were significantly lower in cases (27.9% and 5.5% respectively) than controls (33.1% and 8.0% respectively). Therefore, C-826G3’UTR and T-826A3’UTR haplotypes were associated with reduced risk of ESCC compared to C-826A3’UTR haplotype  (Linkage disequilibrium (LD) analysis showed that NFKBIA -826 C>T and 3’= 0.034) .trend = 0.001). Moreover, in dichotomized analysis, patients with 1-3 risk genotype had 1.67 fold higher risk of ESCC compared to patients with ‘0’ risk genotype   of all four SNPs into new variable according to the number of risk genotypes, ranging from 0-4 . A significant dose dependent risk of ESCC was observed with combined risk genotype of all four SNPs (P= 0.005) . Role of= 0.005) .TNFA -308 polymorphism was the best one factor model and TNFA -308 and NFKBIA 3’UTR were the best model for two factors and TNFA-308, NFKBIA -826, NFKBIA 3'UTR polymorphisms as best model for three factors. However, P value of permutation testing was significant only for three factor model   . Thus, tTNFA, NFKB1 and NFKBIA  with risk and prognosis of ESCC in northern Indian population. We observed that TNFA-308 G>A polymorphism was associated with enhanced risk of ESCC especially in females and in patients with regional lymph node involvements. No independent role of either NFKB1 -94ATTG ins/del or NFKBIA (-826 C>T and 3’UTR A>G) polymorphisms in ESCC susceptibility was found, however, two haplotypes of NFKBIA polymorphisms (C-826 G3’UTR and T-826 A3’UTR) seem to have protective role in ESCC. MDR analysis showed TNFA -308, NFKBIA -826 and NFKBIA 3’UTR polymorphisms as better predictor for risk of ESCC. Moreover, when we pooled risk genotype of all the polymorphisms, a significant increased risk of ESCC was observed in subjects with ≥1 risk genotypes compared to subjects with ‘0’ risk genotype. Inter-individual variation in ESCC may be partly attributed to genetic variants in inflammatory and immune-responsive genes ,39. In tTNFA -308 G>A polymorphism in present study was 7.1% which is similar to Hapmap Guajarati Indian (GIH) and Han Chinese (CHB) population (http//hapmap.ncbi.nlm.nih.gov/). We found a significant 1.7 fold increased risk of ESCC with TNFA -308 GA genotype and A allele in comparison to GG genotype to G allele respectively. Literature suggests strong association of TNFA -308 A allele with MHC haplotype HLA-A1-B8-DR3, which is in turn linked with increased production of TNF-α [TNFA-308A allele is stronger transcriptional activator than G allele and production of TNF-α was reported to be higher in monocyte culture of individuals with TNFA-308 GA compared to that with GG genotype [TNFA -308 GA genotype and A allele may be due to higher TNF-α production, which promotes malignant processes through induction of premalignant chemokines, angiogenic mediators, reactive oxygen intermediates and inflammatory mediators [TNFA -308 G>A polymorphism with susceptibility to gastro-esophageal cancer or ESCC [TNFA-308 polymorphism was not in HWE in controls in these studies (PHWE were less than 0.05). For this reason, we could not perform meta-analysis of TNFA -308 polymorphism in esophageal cancer as one of exclusion criteria for studies in meta-analysis is disagreement of polymorphism with HWE in controls. The MAF of of TNF-α ,41. Alsogenotype ,43. Thus or ESCC ,15. HoweNFKB1 i.e., -94ATTG del is extensively explored in various cancers. The deletion allele of the polymorphism abolishes binding site for nuclear protein resulting in reduced promoter activity [NFKB1 polymorphism with carcinoma of urinary bladder, prostate, cervix and nasopharynx [NFKB1 -94ATTG del polymorphism may be due to its cancer specific or population specific nature of association. We also examined association of two functional polymorphisms in NFKBIA (-826C>T and 3’UTRA>G) with ESCC susceptibility, however no significant role in ESCC was observed.  NF-κB is a major transcription regulator of immune response, apoptosis and cell-cycle genes . A four activity . Althougopharynx ,45–47, nopharynx ,49. The TNFA-308 AA genotype was associated with poor prognosis of tumour at gastro-esophageal (GE) junction study [Univariate survival analysis through Kaplan Meier test and Cox regression showed no difference in median survival or death hazard of ESCC patients with any of the selected gene polymorphism. Also, in multivariate analysis, no effect of studied gene polymorphisms in prognosis of ESCC was found. However, presence of metastasis was associated with worse survival outcome of ESCC patients in multivariate analysis, which is well known fact. A previous study showed that on study . This coTNFA-308 GA was associated with increased risk of ESCC in females, in contrast, NFKBIA -826 CT genotype conferred female-specific decreased risk of ESCC. These is sexual dimorphism in immune/inflammatory response and females exhibits more vigorous cellular/ humoral immune reactions compared to males [TNFA-308 A allele may explained by higher TNF-α level in females leading to aggressive inflammatory response. However, these findings  need to be confirmed in larger cohort and should be interpreted cautiously due to low number of female patients in the present study.Stratification based on gender showed that to males . Also, fto males . So, femTNFA-308 GA genotype conferred significant increased risk of regional lymph node involvement in ESCC patients. Similar to this, Kim et al. had shown association of the TNFA -308 polymorphism with lymph node metastasis in gastric cancer patients [ ESCC shows considerable heterogeneity in clinical phenotypes and different genetic factors/pathways are implicated in development of specific esophageal tumour phenotypes ,53. In lpatients . TNFA-308 GA with regional lymph node involvement in ESCC patients, no role of TNFA-308 polymorphism in clinical outcome of ESCC patients was found.Regional lymph node involvement usually associated with poor clinical outcome of the cancer patients. However, in the present study no modification in prognosis of esophageal cancer patients was observed with lymph node status. This may be due to low number  of followed up patients had regional lymph node involvement. So, despite association NFKBIA polymorphisms which showed significant decreased risk of ESCC with C-826G3’UTR and T-826A3’UTR haplotypes. Bioinformatics analysis showed that NFKBIA -826 C>T polymorphism may affect binding of transcription factor like BRAC1/2, TBP and MYB, while 3’UTRA>G polymorphism may affect binding of mir-196a (www.SNPinfo.com). Few studies have also examined the effect of NFKBIA 3’UTRA>G polymorphism on expression of the protein, however, finding are inconsistent. While, the expression of NFKBIA was reported to be higher in peri-tumour tissues from colorectal cancer patients with 3’UTR AA+AG genotypes than those with 3’UTR GG genotype [NFKBIA in melanoma patients with different 3’UTR genotypes [NFKBIA -826 and 3’UTR polymorphisms in ESCC are lacking, however, it is reasonable to assume that differential expression of IκB due to NFKBIA specific haplotypes may result in different NF-κB activation, further leading to differential risk for ESCC.We next carried out the haplotype analysis of two genotype , anotherenotypes . Data deTNFA-308, NFKBIA -826, NFKBIA 3’UTR polymorphisms was predicted to be best model with maximum testing accuracy and CV consistency. Furthermore, when we pooled the risk genotype (with OR>1) of all the polymorphisms, significant increased risk of ESCC of was observed with combined risk genotype in dose responsive manner. Also, individuals with 1-3 risk genotypes were found to have higher risk of ESCC compared to those with ‘0’ risk genotype. However, no role of combined risk genotype on survival outcome of ESCC patients was found. These findings suggest the joint effect of studied gene polymorphisms in susceptibility but not in prognosis of ESCC. Interaction of selected polymorphisms with environmental risk factors were also analyzed, which did not show any significant outcome. This implies that there may be other classes of genes which might show interaction with environmental risk factor for developing risk of ESCC in northern Indian population.Cancer is a multi-genic disease in which single SNP may only have a modest independent effect on disease phenotype and multiple SNPs may provide a more accurate representation of the risk. So we carried a high order interaction analysis to find out the best predictive model for the risk of ESCC. A three factors model The present study has several strengths like well defined set of cases and controls, agreement of genotypic data with HWE in controls, consistencies of MAF of polymorphisms with Hapmap GIH data and adoption of stringent quality control measures. Limitation of study is low sample size in subgroups, absence of qualitative environmental exposure data in cases and short median follow-up in survival analysis. TNFA -308 G>A polymorphism and combined effect of TNFA and NFKBIA gene polymorphisms in susceptibility of ESCC in northern Indian population. However, none of the genetic variants seem to have implications in prognosis of ESCC. In summary, our results suggest independent role of Table S1Details of genotyping methods of TNFA-308 G>A, NFKB1 -94ATTG ins/del and NFKBIA (-826 C>T and 3’UTR A>G) polymorphisms.(DOCX)Click here for additional data file.Table S2Multi Dimensionality Reduction (MDR) analysis of selected gene polymorphisms.(DOCX)Click here for additional data file.


Mycobacterium ulcerans. Its exact transmission mechanism remains unknown. Several arguments indicate a possible role for insects in its transmission. A previous case-control study in the Nyong valley region in central Cameroon showed an unexpected association between bed net use and protection against Buruli ulcer. We investigated whether this association persisted in a newly discovered endemic Buruli ulcer focus in Bankim, northwestern Cameroon.Buruli ulcer is an infectious disease involving the skin, caused by We conducted a case-control study on 77 Buruli ulcer cases and 153 age-, gender- and village-matched controls. Participants were interviewed about their activities and habits. Multivariate conditional logistic regression analysis identified systematic use of a bed net (Odds-Ratio (OR) = 0.4, 95% Confidence Interval [95%CI] = [0.2–0.9], p-value (p) = 0.04), cleansing wounds with soap  and growing cassava  as independent protective factors. Independent risk factors were bathing in the Mbam River  and reporting scratch lesions after insect bites . The proportion of cases that could be prevented by systematic bed net use was 32%, and by adequate wound care was 34%.M. ulcerans infection should now be investigated.Our study confirms that two previously identified factors, adequate wound care and bed net use, significantly decreased the risk of Buruli ulcer. These associations withstand generalization to different geographic, climatic and epidemiologic settings. Involvement of insects in the household environment, and the relationship between wound hygiene and  Mycobacterium ulcerans is the causative agent of Buruli ulcer disease, which causes skin wounds and often results in disabilities. The transmission of M. ulcerans remains unknown. Environmental and biological studies have gathered evidence that insects could play a role in M. ulcerans circulation. A case-control study performed in central Cameroon in 2007 unexpectedly illuminated an association between bed net use and a decreased risk of Buruli ulcer. As this result suggested a potential domestic transmission involving insects, we set up a new study to investigate whether this association existed in Bankim, a newly discovered Buruli ulcer endemic site in northwestern Cameroon. Our results confirm the protective effect of bed nets in this region, despite very different population, environment, and climate factors. They also confirm the role of good hygienic practices, a protective factor repeatedly identified in previous studies. These repeated associations now warrant further research on a possible domestic or peri-domestic transmission of the disease, involving local water collections and possibly insects. Over the last five years, Buruli ulcer (BU) care has been greatly improved by implementation of antimicrobial therapy and more effective diagnosis methods. However, much about the transmission of this devastating skin disease remains to be learnt. The incidence of BU disease is still on the rise , and its emergence or increasing incidence have been linked with environmental modifications in rural areas, such as construction of dams or irrigation systems Studies aimed at identifying BU risk factors have repeatedly shown an association between the disease and activities such as wading In 2006, a case-control study performed in Akonolinga, in the endemic region of the Nyong valley in central Cameroon, showed for the first time that the use of a bed net was associated with protection against BU Mycobacterium ulcerans. Several water bugs (aquatic Hemipterans) species M. ulcerans in endemic areas. Furthermore, saliva of Hemipterans captured in the environment has been shown to harbor M. ulcerans, and the first M. ulcerans culture from an environmental sample was obtained from a semi-aquatic Hemipteran This finding is consistent with a possible role of insects in the transmission of Laboratory experimental infection of mice bitten by infected water bugs provides important biological support for their involvement Protection against insect bites using a bed net could present a simple, easily implementable means of BU prevention.We designed this replication study The Bankim BU focus shows several specificities compared to the endemic region of the Nyong river basin in central Cameroon. First, the disease was unknown in the area until recent years . It was reported 15 years after construction of a dam on the Mape River which caused major modifications in the environment and relocations of populations and activities after submersion of villages and farmland. Secondly, the region's geographic, climatic and demographic context differs from that of the forested Nyong River basin. Bankim is located in a transitional zone between forest and savanna and at the foot of the Mambila Mountains; its tropical seasonality is more contrasted than in the equatorial south; and the region is populated by a rich diversity of ethnic groups with specific cultural features. Due to its ecological, demographic and ethnic heterogeneity, Bankim district represented an adequate setting to challenge the hypothesis of bed net use as a protective factor against BU.Realizing a study in Bankim provided an opportunity to document the other risk factors for BU in this newly discovered focus. It also contributed to raising awareness of BU among Bankim's diverse populations, who considered the disease to result from occult origins and who frequently under-report it.We conducted a matched case-control study in the district of Bankim, Adamaoua region, Cameroon.A probable case of BU was defined as a patient presenting with active or inactive BU disease, diagnosed between 1 January 2007 and 15 August 2009 by trained health personnel  and treated according to WHO recommendations at the district hospital in Bankim.M. ulcerans infection, proved by identification of acid-fast bacilli in stained smears from swabs taken from lesions, amplification of IS2404 by polymerase chain reaction, or both. Analyses were performed at the Mycobacteria Reference Laboratory in Centre Pasteur du Cameroun, Yaoundé, as described previously A confirmed case was defined as a probable case with laboratory evidence of Cases originating from outside the health district were not included in the study. Cases reporting having lived with lesions for 10 years or more were not included, since reporting of exposures before the disease would have been inaccurate and comparison with activities of controls over the last year would have been irrelevant.Two kinds of controls were enrolled.An eligible community control was defined as a person without signs or symptoms of active or inactive BU and who was not related to a BU case . Two controls matching cases for age (+/−2 years), sex, and village were randomly selected. Census data was collected in June 2009 by the district “community health agents” during activities of the National Onchocerciasis Control Program. For each village, a list presenting the name, gender and age of each inhabitant was obtained. Using this list, we assigned a number to each person in the village matching a case for age and gender, and then drew randomly five numbers among all numbers assigned. Controls were contacted in the order they were drawn, received preliminary information from the village “community health agent”, and were given an appointment for an interview. If this person failed to show up at two successive appointments, we visited the next control on the list until two controls were interviewed. In three instances where census data were not available, we selected as controls the first persons matching age and sex criteria and living in a randomly chosen house in the village.An eligible familial control was defined as a healthy member of the family closest in age to the case, within the same age class . Siblings of the case were preferentially included, but more distantly related people living in the same household could be included in the absence of siblings.Study enrollment was voluntary. Information was provided first to the village population then individually to the participants. Written informed consent was obtained from case patients and control subjects or from their parents or guardians if participants were younger than 18 years. All BU case patients had received or were currently receiving free treatment for BU at the district hospital, a local clinic or the reference hospital for BU in Ayos. The study protocol was approved by the National Ethics Committee and the Cameroon Ministry of Public Health.The sample size computed for this matched case-control study required 71 groups of one case and two community controls to document an odds-ratio of 0.4 or less as statistically significant, with the following assumptions: proportion using bed nets in the control population = 0.5  were provided by the same interpreter when needed. Cases were interviewed about their activities during the year before the onset of disease, while controls were asked to report their activity during the previous year.Data were analyzed using STATA version 10.0 . Univariate analysis was performed using conditional logistic regression to calculate odds-ratio (OR) and 95% confidence intervals (95%CI). Missing values for a given variable were not included in the univariate analysis of this variable. Variables with p-values lower than 0.25 in univariate analysis were included in a multivariate model.Multivariate analysis was performed on these variables using multivariable conditional logistic regression after a first step of intra-exposure category selection. Independently associated variables among an exposure category were in a second step introduced in a multi-exposure category model. At each of the two steps, a stepwise backwards procedure was used to remove from the model the variable with the highest p-value until all p-values were less than 0.05. This procedure (clogit) compared nested conditional logistic regression models by a likelihood ratio test.In the final model, the “interviewer” effect was assessed by creating a variable “interviewer” coded “0” or “1”, and by testing whether odds ratios of the significant associations of the final model were “modified” when adding an interaction term between each variable of the final model and the variable “interviewer”. Interactions between variables of the final model and age or gender variables were tested using the same method. Age was recoded in 4 categories according to quartiles .Preventive fraction was defined as the proportion of cases that could be avoided by systematic exposition of the population to a given protective factor. For instance, the preventive fraction for adequate wound hygiene is the proportion of Buruli ulcer cases that would be avoided if every person in the population cleansed their wounds with soap regularly. Preventive fraction was calculated for each protective factor using the odds ratio estimated in the final model according to the formula developed by Miettinen Preventive Fraction = [P(having disease/unexposed)−P(having disease)]/P(having disease/unexposed) = P(Exposed)×(1−OddsRatio).Of the 195 probable Buruli ulcer cases recorded in Bankim district hospital since January 2007, 100 cases were contacted, 88 were questioned, and 77 retained for analysis. Only one BU patient refused to participate. Cases were not included or not considered for analysis if they reported having suffered from BU for ten years or longer (n = 11), if one relative  was already included in the study (n = 5) or if their distant location did not allow us to investigate community controls (n = 6). Details of the inclusion process are provided in Community controls were randomly selected in the population census for 74 cases. For 3 cases living in villages for which census data were not available, controls were included from randomly selected houses. The main reasons for controls not participating were long-term or repeated absence , refusal , unable to be found , or related to a case .The main analysis was performed on 77 cases (72 probable and 5 laboratory confirmed cases) matched with 153 community controls. Out of 77 cases analyzed, 76 were matched with two controls and one case was matched with one control only.Familial controls were recruited when available, i.e. mainly for children. Case-familial control analysis was performed on 37 cases and 49 controls. These were distributed as follows: 12 groups of one case and two controls and 25 groups of one case and one control. Among controls, 42 were siblings, 5 were other relatives  and two were unrelated children living in the same household.Most patients were healed, except for 16, who presented with lesions still not scarred . Five caAbout as many cases were men (n = 40) as women (n = 37). Median age of analyzed cases was 14 years (Inter-quartile Range (IQR) = [10–36.5]).Lesions classically presented on lower , and upper limbs . Only 3 patients presented with multiple lesions, and 8 (10%) presented with head or trunk lesions. Median (IQR) time from onset of disease to day of interview was 17 (9–26) months, ranging from 15 days to 7 years.The majority of patients did not associate their disease with any particular circumstance, but 9 linked it to a wound and 17 to insect bites.The overall population of the study, presented in Adults principally engaged in farming activities. Most children between 6 and 12 years old reported going to school (94%), and a majority of them participated in farming during weekends and holidays or during seasons when additional workforce was required (85%). Education level was low with few persons over 12 years of age having secondary education.Neither ethnic group, activity nor education level was significantly associated with BU . A histoChrysops genus (Tabanidae), but the association was not significant. BU was significantly associated with reporting history of small skin lesions due to scratching after an insect bite . Mosquito coils were seldom used and no association with BU was found.Exposure to insects was assessed by questioning about specific insects bites . Cases rWhen compared to never using a bed net, systematic bed net use was associated with protection from BU and occasional use was associated with an increased risk . Most bed nets had been bought from the local market and were not insecticide-treated. Net insecticide treatment did not appear associated with better protection, nor did the absence of holes.Exposure to water was assessed for various activities . FishingBathing activities were separated into bathing for hygiene and for leisure. Bathing for hygiene was statistically associated with an increased risk of BU when baths were taken in the Mbam, a river flowing south from the district . Bathing in the barrage lake or other water bodies was not associated with a statistically significant change in the risk of disease. Bathing for leisure was a common activity for nearly all children and did not significantly increase risk.Domestic water supply and clothes washing activities were investigated, but no significant difference in exposure was found between cases and controls. Water sources were mainly rivers, streams or wells. Protected water sources were limited to urban compounds and not necessarily used for all activities. Whether unprotected water sources were running or stagnant was difficult to assess, but no statistically significant association was found between BU and declared characteristics of the water source.Not wearing shoes during domestic water supply or clothes washing was significantly associated with an increased risk of BU . Type of clothing or shoes worn during any other water-related activity was not associated with a significant risk of BU.Almost all cases and controls participated in farming activities . YoungesCases were more likely than controls to have a longer walking time to their fields and to sleep there. They more frequently reported cultivating areas that flooded during the rainy season. Cases also more often declared having a garden or a vegetable patch near their houses, and watering these plots was associated with a significantly increased risk.Clothing worn during farming activities was not associated with a risk of BU.Wounds were frequent in the population, and were usually acquired during farming activities . ControlSeveral wound treatments were associated with a change in BU risk: using alcohol or soap to cleanse the wound were protective practices, while using no treatment led to an increased risk of disease. Rubbing leaves  or applying ground tablets or ointment bought on the market was not significantly associated with the risk of BU disease. Dressing the wound was a protective practice when the bandage - either a piece of cloth or adhesive bandage - was regularly changed.For multivariate analysis, the main exposure “bed net use” was recoded in two categories, “Systematic bed net use Yes/No” by pooling the two categories “Never” and “Rare to often”, which were both associated with an increased risk of disease in the univariate analysis .Independent factors associated with an increased risk of BU were bathing for hygiene in the Mbam River  and reporting small scratch wounds after insect bites . Factors independently associated with a decreased risk were systematic use of a bed net , use of soap to cleanse wounds  and growing cassava .No significant interaction was found in the final model between variables and a variable “interviewer”. No significant interaction was found between these variables and age category or gender. Detailed results of variable selection process are provided in supplementary Univariate analysis showed a few associations that did not reach significance level . Of noteIf we assume that the relationship between a protective factor and risk decrease is truly causal, we can calculate the proportion of cases in the general population (represented by the controls) prevented by this protective factor, based on the relative risk and the frequency of the factor in the general population .The preventive fraction for systematic bed net use was 32% . Likewise, the fraction of cases prevented by adequate wound hygiene practices (using soap to cleanse wounds) was 34% .This study identified practices that demonstrate statistically significant associations with Buruli ulcer in a newly endemic area of northwestern Cameroon. Systematic bed net use, adequate wound hygiene practices, and growing cassava were associated with a decreased risk of disease. Bathing for hygiene in the Mbam River was associated with an increased risk, as well as reporting frequent occurrence of scratch wounds following insect bites.The chosen setting, Bankim district in the Adamaoua region, contrasts with the previous study site. Akonolinga, the previous study site, was characterized by a relative homogeneity: 80% of the population belonged to the same ethnic group, the region was largely forested and water-related activities were centered on a major river, the Nyong Our rigorous sampling method, which used census data and repeated appointments with controls, allowed us to capture accurately the heterogeneities of populations and activities in Bankim and to minimize control recruitment associated biases Similar to case-control studies on BU, the present study has several limits inherent in its methodology and context. First, even if we are confident that our sampling method prevented most sampling biases, memory bias could have occurred: cases were interviewed on their activities during the period preceding the onset of disease and could therefore remember them differently from controls, who were interviewed on their activities over the previous year. However, we included mainly cases with disease diagnoses during the two previous years. This ensured that cases and controls would recall their activities on similar time scales and minimized differential bias. Furthermore, having suffered Buruli ulcer disease could have influenced cases' answers to questions, such as the occurrence of scratch wound as discussed below , suggesting that it was not confounded by socio-economic status. A larger proportion of adults was included in the present study which made it more difficult to recruit siblings as familial controls. Statistical power is consequently low in the family-matched case-control study. However, use of a bed net still presents a protective effect in this sub-study, which, although not significant, has the same magnitude (OR [95%CI] = 0.4 [0.1–1.4]) as in the community-matched study (OR [95%CI] = 0.4 [0.2–0.9]). While socio-economical status might have confounded the association in the community-matched study, if for instance, participants using bed nets were less likely to have activities exposing them to Here we demonstrate that the association between bed net use and protection against BU withstands generalization in a different climatic, geographic, environmental, and ethnic context. This replicative study greatly increases the positive predictive value of the association between bed net use and decreased risk of BU Few epidemiological studies have investigated the possible role of a bed net as a protective factor against BU in Africa. In Ghana, no significant association was found between bed net use and protection against BU, but the proportion of bed net users in the general population was low (25%), and the 116 pairs of cases and controls only yielded a power 67% to show an OR of 0.4 or less The preventive fraction for systematic bed net use was 32% in this study, and reached 43% in the previous study performed in Akonolinga M. ulcerans by an insect vector. This hypothesis is consistent with several results from environmental studies. In Australia, M. ulcerans infection is now considered a zoonosis transmitted from possum to human by mosquitoes The protective effect of bed nets could result from two different mechanisms. First, using a bed net could protect from direct inoculation of M. ulcerans to enter the skin when in contact with contaminated environment. Occurrence of scratch wounds following insect bites was indeed associated with an increased risk of disease in our study. This factor was statistically independent of bed net use as shown by the persistence of both variables in the final model. This result suggests that both risk factors may act independently, some insect bites being not preventable by bed nets, for instance those occurring at day time.Secondly, bed nets could provide a protection by preventing unspecific insect bites. These bites can lead to small lesions after being scratched which would enable M. ulcerans in Aedes and Anopheles mosquitoes which are important in Africa. Studies on distribution, abundance and M. ulcerans positivity of water bugs should not be restricted to the aquatic environment and seek these insects in the domestic environment.The association between bed net use and protection against BU underlines the need to broaden environmental studies to aerial insects and to the domestic environment. It would be relevant to assess the presence of The strongest protective association was linked to using soap to cleanse wounds. This variable summed up others that implied practices of proper wound care: use of alcohol and frequent bandage changing were also associated with protection, and usually concerned the same people. This association of a decreased risk of Buruli ulcer and good hygiene practices has been found in Ghana M. ulcerans, other transmission mechanisms are likely to coexist that involve wounds as a port of entry, such as contamination of open wounds by water, or direct inoculation of M. ulcerans by a wounding object.This also indicates that while some insects may transmit Several mechanisms could explain the decreased risk of disease associated with wound hygiene. Clean wounds heal faster and could be less likely to become a port of entry for the microbe. Frequent wound cleansing could also prevent the survival of the microbe in the skin: since the initial mycobacterial inoculum is expected to be low  and short clothing related associations (infrequently used for farm work or fishing). Bankim environment's heterogeneity probably prevented us from identifying major water sources of exposure: there is a large number of water bodies, and people change both water-related activities and locations throughout the year because these water sources dry up during the dry season. This may explain why in this study, four behavioral factors and only one location-dependent factor had statistically significant associations with BU in the final model.Through this study, we have demonstrated that the association between bed net use and protection against BU, found in a previous case-control study in Akonolinga, can be generalized to another climatic, geographical, environmental and ethnic context. We also identified good wound hygiene as a key protective factor, as reported in several previous studies.M. ulcerans, by direct (skin lesions) or indirect (insect bites) contamination involving peri-domestic water collections. We believe that it is now of critical interest to confirm or to rule out the domestic vector hypothesis by investigating insects in the domestic and peri-domestic environment of BU endemic foci. Such study would also help to determine if implementation of bed net protection would help prevent BU transmission.These findings support the hypothesis of a domestic or peri-domestic transmission of M. ulcerans transmission merits investigation in greater detail, so as to promote specific hygienic practices through prevention strategies.Furthermore, good hygiene appears again as a key protective factor: using soap to cleanse wounds was found protective, while hygiene baths in the Mbam River increased the risk of disease. Hygienic water sources and behaviors appear to be connected. The link between hygiene and M. ulcerans infection should be encouraged so that meaningful and effective prevention strategies can be identified and proposed.Finally, we believe that while clinical research is making progress in developing oral treatment of Buruli ulcer Figure S1Map of the Bankim area presenting the number of cases per village from January 2007 to August 2009.(DOC)Click here for additional data file.Table S1Characteristics of the 77 analyzed cases.(DOC)Click here for additional data file.Table S2Case-family matched control study analysis. Univariate and multivariate analysis results of risk factor analysis in Bankim, Cameroon, 2007–2009.(DOC)Click here for additional data file.Text S1Details on multivariate analysis  for case-community matched control analysis of risk factors for Buruli ulcer in Bankim, Cameroon, 2007–2009.(DOC)Click here for additional data file.Protocol S1Trial protocol.(DOC)Click here for additional data file.Checklist S1CONSORT checklist.(DOC)Click here for additional data file.


XRCC1) Arg399Gln polymorphisms with susceptibility to squamous cell carcinoma of the head and neck (HNSCC). However, the results were conflictive rather than conclusive. The purpose of this study was to clarify the association of XRCC1 Arg399Gln variants with HNSCC risk.Epidemiologic studies have reported the association of X-ray repair cross-complementary group 1  with 95% confidence intervals (CI) was computed to estimate the strength association.XRCC1 Arg399Gln polymorphisms with HNSCC risk in total population  based on 18 studies including 3917 cases and 4560 controls. In subgroup analyses, we observed an increased risk of XRCC1 399 Arg/Gln genotype for HNSCC in Caucasians  and Gln/Gln genotype for larynx squamous cell carcinoma . We did not observe any association between XRCC1 Arg399Gln variants and HNSCC risk in additional subgroup analyses.Overall, we did not observe any association of XRCC1 Arg399Gln variants may contribute to HNSCC risk among Caucasians and to the risk of larynx squamous cell carcinoma. Further, well-designed studies with larger sample sizes are required to verify our findings.The results from this present meta-analysis suggest that  The global incidence of squamous cell carcinoma of the head and neck (HNSCC) is 540,000 per year, with an annual mortality rate of 271,000. HNSCC is a malignant disease of the upper aerodigestive tract, including oral, pharyngeal and laryngeal regions. HNSCC occurs most commonly in men between the sixth and seventh decade of life. Epidemiologic data have shown that tobacco smoking and alcohol consumptions are the main etiological factors in the carcinogenesis of squamous cell carcinoma of the head and neck Although a majority of people are exposed to such risk factors, only a small fraction of them develop HNSCC XRCC1 Arg399Gln polymorphisms and HNSCC risk XRCC1 Arg399Gln polymorphisms with HNSCC risk in 2007 and did not observe any significant association based on 7 published studies. Ever since, several new studies have provided additional data on the association between XRCC1 Arg399Gln polymorphisms and HNSCC risk. In order to address a more precise estimation of this relationship, a meta-analysis including a total of 18 studies was performed, which may provide the more comprehensive evidence for the association of XRCC1 Arg399Gln variants with HNSCC risk.XRCC1 is a major DNA repair gene involved in base excision repair (BER) and single-strand breaks (SSBs) repair. XRCC1 interacts strongly with Poly [ADP-ribose] polymerase 1 (PARP1), which recognizes SSBs, and LIGIII that seals SSBs and BER intermediates XRCC1”. Additional studies were identified using the reference lists of the selected papers. The ending date of publication search was March 1, 2013.Systematic searches in Pubmed/Medline, Elsevier, Science Direct, Chinese National Knowledge Infrastructure (CNKI) and Chinese Biomedical Literature Database were performed using the following search terms: “head and neck cancer” or “oral cancer” or “larynx cancer” or “pharynx cancer” and “polymorphism” and “XRCC1 Arg399Gln polymorphisms and HNSCC risk; (2) Case-control studies or cohort studies; (3) Sufficient data were used for estimating OR with 95% CI; (4) When more than one article was identified for the same study population, we included the publication containing more information. The exclusion criteria were (1) studies on nasopharyngeal cancer; (2) studies that could not offer the number of cases and controls or other essential information; (3) reviews or studies with overlapping patient populations; (4) studies without histologically confirmed information for HNSCC.The following criteria were used for study selection: (1) The papers should evaluate the association between XRCC1 Arg399Gln polymorphisms and head and neck cancer risk. We reviewed all papers in accordance with the criteria listed, above; seven studies without histologically confirmed information for HNSCC, three overlapping studies and two reviews were excluded. At last, 18 original articles that reported the association between XRCC1 Arg399Gln polymorphisms and HNSCC risk were determined to be eligible to enter our study  and STATA10.0 software package . All the tests were two-sided, a P value of HWE. There were a total of 18 studies with 3917 cases and 4560 controls concerning the XRCC1 Arg399Gln polymorphisms related to HNSCC risk.A database was established according to the extracted information from all eligible articles. Essential information was listed in XRCC1 codon 399 Gln/Gln vs. Arg/Arg, Arg/Gln vs. Arg/Arg and Gln/Gln+Arg/Gln vs. Arg/Arg was analyzed for 18 identified studies. The results showed that XRCC1 codon 399 Gln/Gln vs. Arg/Arg, Arg/Gln vs. Arg/Arg and Gln/Gln+Arg/Gln vs. Arg/Arg for Caucasians and oral squamous cell carcinoma, Gln/Gln vs. Arg/Arg for healthy population-based control and larynx squamous cell carcinoma, and Arg/Gln vs. Arg/Arg for hospital-based control and healthy population-based control had no heterogeneity with a P value ≥0.05, therefore, we analyzed the summary odds ratios for them with a fixed-effects model. Random-effects model was used to analyze the summary ORs for the rest.The heterogeneity of XRCC1 Arg399Gln polymorphisms related to HNSCC risk on the basis of 3917 cases and 4560 controls. We did not observe any association of XRCC1 Arg399Gln polymorphisms with HNSCC risk in the total population  and Gln/Gln genotype for larynx squamous cell carcinoma . We did not observe any association between XRCC1 Arg399Gln variants and HNSCC risk in additional subgroup analyses  based oanalyses .Publication bias was assessed by funnel plot, the shape of the funnel plot seemed to be approximately symmetrical , 5, 6. TSensitivity analyses were conducted to determine the influence of the individual dataset on the summary ORs by sequential removal of each eligible study. The overall effects were not altered when the studies were homogenous for Gln/Gln vs. Arg/Arg, Arg/Gln vs. Arg/Arg and Gln/Gln+Arg/Gln vs. Arg/Arg among total population by removing some eligible studies , S3, S4.http://www.ncbi.nlm.nih.gov/SNP), among them, rs25487 in XRCC1 codon 399 of exon 10 was the most extensively studied polymorphic site. Recently, meta-analysis studies have reported that XRCC1 Arg399Gln variant was associated with the risk of certain cancers, such as breast cancer XRCC1 Arg399Gln and the risk of squamous cell carcinoma of the head and neck. We also estimated the possible effect modifications by source of control, ethnicity of subjects, tumor site and HWE in control. In summary, we did not observe an association of XRCC1 Arg399Gln polymorphisms with HNSCC risk in the total population. Our findings are consistent with the previous meta-analysis study XRCC1 is an important component of the base excision repair system, which is the predominant DNA repair pathway for small base lesions resulting from oxidation and alkylation damage XRCC1 Arg399Gln polymorphisms with oral squamous cell carcinoma. We did not perform subgroup analyses on other tumor sites, owing to unavailable data.HNSCC includes tumors from different sites including oral, pharynx and larynx region, risk factors for these cancers may be different. For example, oral cavity and laryngeal cancers are mainly associated with tobacco use and alcohol consumption, while oropharyngeal cancers are principally related to viral infection, such as human papillomavirus (HPV) XRCC1 399 Gln allele variant is significantly different among different ethnic population. Allele frequency patterns of XRCC1 Arg399Gln polymorphism vary greatly among major ethnic groups. The frequency of Gln allele was more than 0.3 in Caucasians XRCC1 Arg399Gln polymorphisms with HNSCC risk among Caucasians, but not among Asian population. Different ethnicities cancer susceptibility associated with the XRCC1 Arg399Gln polymorphisms was also observed in previous meta-analyses of lung cancer and breast cancer Considering that the frequency of the It is widely acknowledged that deviation from HWE may point to methodological weaknesses, such as biased selection of subjects or genotyping errors. The results of genetic association studies might be spurious if the distribution frequency of genotypes in the control groups was not in agreement with HWE XRCC1 Arg399Gln polymorphisms and HNSCC risk neither in hospital-based control nor in healthy population-based control. Thirdly, the summary odds ratios were based on individual unadjusted estimates, while a more precise analysis might be performed if detailed individual data were available, which could allow for an adjusted estimation by sex, age, tobacco use, alcohol consumption and environment factors. In addition, the study number was limited and the sample size was relatively small in subgroup analyses, as a consequence, our estimates of the association in subgroup analyses might have occurred by chance. Thus, the results must be interpreted with caution.This meta-analysis should be interpreted within the context of its limitations. First, only published articles were included in this study. Therefore, publication bias is very likely to occur. To address this issue, Egger’s test and Begg’s test were conducted. Our results indicated that the likelihood of key publication bias might not be present in this meta-analysis. Secondly, each study had different eligibility criteria for subjects and different source of controls, which should be taken into account while interpreting the combined estimates. When subgroup analysis was performed by source of control, we observed any association between XRCC1 Arg399Gln variants appear not to be a risk factor of HNSCC in the total population. However, XRCC1 Arg399Gln variants might be a potential risk factor for HNSCC among Caucasians and for larynx squamous cell carcinoma. Large scale studies with the pooling of individual study data should be taken into account in the future studies to verify the results from this current meta-analysis.In conclusion, this systematic review demonstrates that Figure S1Flow diagram.(TIF)Click here for additional data file.Figure S2Sensitivity analysis for Gln/Gln vs. Arg/Arg.(TIF)Click here for additional data file.Figure S3Sensitivity analysis for Arg/Gln vs. Arg/Arg.(TIF)Click here for additional data file.Figure S4Sensitivity analysis for Gln/Gln+Arg/Gln vs. Arg/Arg.(TIF)Click here for additional data file.Checklist S1PRISMA 2009 Checklist.(DOC)Click here for additional data file.


CYP26B1 rs2241057 polymorphism with TaqMan® SNP Genotyping Assay followed by allelic discrimination analysis. A higher frequency of patients homozygous for the major (T) allele was associated with CD but not UC compared to the frequency found in healthy controls. A significant association between the major allele and non-stricturing, non-penetrating phenotype was evident for CD. However, the observed associations reached borderline significance only, after correcting for multiple testing. We suggest that homozygous carriers of the major (T) allele, relative to homozygous carriers of the minor (C) allele, of the CYP26B1 polymorphism rs2241057 may have an increased risk for the development of CD, which possibly may be due to elevated levels of retinoic acid. Our data may support the role of Vitamin A in the pathophysiology of CD, but the exact mechanisms remain to be elucidated.Several studies suggest that Vitamin A may be involved in the pathogenesis of inflammatory bowel disease (IBD), but the mechanism is still unknown. Cytochrome P450 26 B1 (CYP26B1) is involved in the degradation of retinoic acid and the polymorphism rs2241057 has an elevated catabolic function of retinoic acid, why we hypothesized that the rs2241057 polymorphism may affect the risk of Crohn’s disease (CD) and Ulcerative Colitis (UC). DNA from 1378 IBD patients, divided into 871 patients with CD and 507 with UC, and 1205 healthy controls collected at Örebro University Hospital and Karolinska University Hospital were analyzed for the  In the first step, vitamin A is reversibly oxidized to retinal by retinol dehydrogenases (RDHs), while step two irreversibly transforms retinal into atRA via retinal dehydrogenases (RALDHs) CYP) genes, which is important for the elimination of atRA and thereby crucial for the control of the atRA homeostasis in the cell, both in the embryonic life as well as in the adult life + cells in SAMP1/YP mice Inflammatory bowel disease (IBD) is the collective name of several chronic relapsing inflammatory disorders of the gastrointestinal tract, dominated by Crohn’s disease (CD) and ulcerative colitis (UC). These diseases are characterized by altered intestinal immune response against gut microbiota in genetically susceptible individuals. The precise pathogenesis is, however, still unclear. Previous studies have indicated on a relationship between the vitamin A analogue isotretinoin and the development of IBD CYP26B1 gene possessed enhanced retinoid catabolizing capacity CYP26B1 gene may be involved in the pathogenesis of IBD. In the present study we aimed to investigate this polymorphism as a risk factor for the development of CD and UC, respectively, and to assess possible genotype-phenotype associations in a case-control study.Previously, a study from our group showed that the minor allele (C) of the rs2241057 polymorphism encoding a Leu to Ser amino acid shift in position 264 in the Ethical approval was obtained from the regional ethical review boards of Karolinska Institutet, Stockholm, Sweden and Uppsala University, Uppsala Sweden. Written informed consent was obtained from IBD patients and verbal informed consent from healthy blood donors recruited at the Blood Bank of the Örebro University Hospital in accordance with the ethical approval. At arrival at the Blood Centre, blood donors were provided with written information, verbally informed about the study, and given the possibility to ask questions about the study. Exclusion criteria were history of any chronic gastrointestinal disease, including IBD. If a blood donor verbally approved to participate in the study, a blood sample was collected for the genetic analyses. This procedure was approved by the regional ethical board, only age and sex of the blood donors were recorded and there is no way to trace back identities of the included individuals. Furthermore, the ethical committee approved that the consent was not recorded in the medical notes, since the enrolment was anonymous. The ethical considerations for the study followed the principles of the Declaration of Helsinki.In total, 1378 Swedish patients with IBD, 871 with CD and 507 with UC, were recruited at Örebro University Hospital, Örebro and the Karolinska University Hospital, Stockholm. Diagnosis was based on standard clinical, endoscopic, radiologic and histologic criteria CYP26B1 gene, the TaqMan® SNP Genotyping Assay C_ _15872328_10  was used. The amplification mixtures contained 40 ng DNA, 1xTaqMan® Genotype PCR Master Mix (Applied Biosystems), 1xTaqMan® SNP Genotyping Assay (Applied Biosystems) including allele specific probe and primers labeled with the two allele specific fluorescent reporter dyes VIC  and FAM , and Milli-Q water in a 10μl reaction. The PCR reactions were performed on 7900HT Fast Real-Time PCR System (Applied Biosystems) using 96 well plates. The PCR profile was as follows: 10 min at 95°C and 40 cycles of 15 s at 95°C followed by 60 s at 60°C. Six negative controls were included on each plate. The amplification was followed by post-read allelic discrimination analysis.DNA was extracted from whole blood using QIAamp DNA Blood Maxi Kit  according to the manufacturer’s instructions. For the genotyping of the SNP rs2241057 polymorphism in the http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=2241057), and an odds ratio of three in cases vs. controls, 656 patients with CD and UC, respectively, as well as 656 controls would be required to obtain a statistical power of 80% and a statistical significance level of 5%.The rs2241057 polymorphism was tested for Hardy-Weinberg equilibrium. Based on the assumptions of a CC genotype prevalence of 3.5% in the background population  and 95% confidence interval (CI) as well as false discovery rate (FDR) corrected P-values, due to analyses of three groups, i.e. CD, UC and IBD. A P-value ≤ 0.05 was regarded as statistically significant.Statistical analysis was performed with the SPSS , Epi Info  and STATA release 11  packages. Associations between categorical variable were assessed with Chi-square test or the Fisher’s exact test (two tailed) when appropriate. The data were presented as uncorrected CYP26B1 rs2241057 polymorphism was observed in patients with CD compared with healthy controls for the CYP26B1 rs2241057 polymorphism  allele compared to the minor (C) allele in the vs. the CC genotype was observed in this subgroup of patients , compared to homozygous carriers of the minor (C) allele  allele seems to be a possible risk factor for the development of CD. We have previously shown that the minor (C) allele catabolizes retinoic acid more efficient than the major allele CYP26B1 rs2241057 is probably associated with higher levels of retinoic acid, due to the lower catabolic activity, which may result in an increased risk of CD. Our results are supported by the observed risk of IBD in patients treated with the vitamin A analogue isotretinoin (13-cis-retinoic acid) for severe acne IBD, including CD and UC, is an idiopathic condition with chronic inflammation of the gut. Several pieces of evidence support the role of the Vitamin A in the pathophysiology of IBD. AtRA, the most bioactive metabolite of Vitamin A/retinol, is mainly regulated by CYP26 proteins. Our findings of a possible association between the et al. showed that Vitamin A deficiency induces colonic inflammation in vitro in murine colitis et al. showed that both limited and excessive vitamin A conditions induced distinct FoxP3(+) T-cell subsets, and both ameliorated the intestinal inflammation in mice Despite increasing data, the role of retinoids in IBD is still controversial. Contradictory to our findings, Reifen h1-type T-cell mediated inflammation, but in more recent years the role of IL-17 secreting Th17 cells have been discovered h17 cells CYP26B1 rs2241057 polymorphism were significant for CD patients with inflammatory behavior or young age at diagnosis, and not for patients with the other sub-phenotypes of CD. It can be speculated if the observed association between the CYP26B1 rs2241057 polymorphism and CD is mediated by changes in the atRA metabolism and thereby altered induction and homing of Th17 cells within the IL-23/Th17 pathway.CD has historically been associated with a TThe fact that our findings could not be confirmed when comparing allele frequencies is a limitation of the study. However, based on the biologic driven hypothesis and previous clinical observations in vitamin A treated IBD patients, we aimed to detect differences in genotype. A study design powered to demonstrate differences in allele frequencies would require an enormous number of cases and controls. Our findings are also limited by the observation that the associations in CD were not significant, after correcting for analyses of three subgroups, i.e. CD, UC and the entire IBD cohort. However, the study was not powered to detect significant differences after correction for analyses of CD, UC and the total IBD population. It can not be excluded that absence of significant associations in UC is explained by the lower number of enrolled patients with UC, although some genetic variants seem to be disease specific and others are shared by both CD and UC CYP26B1 polymorphism rs2241057 and CD. Since the CYP26B1 gene is one of the major regulators of vitamin A our data supports the role of vitamin A in CD. Further replication efforts and mechanistic studies exploring the influence of vitamin A and CYP26B1 in the pathogenesis of CD are warranted.In conclusion, we observed a possible association between homozygous carriers of the major (T) allele of the Table S1Genotype frequencies of the polymorphism rs2241057 in the CYP26B1 gene for patients with Ulcerative colitis and healthy controls, displayed for sub phenotypes and clinical features.(DOCX)Click here for additional data file.Table S2Allele frequencies of the polymorphism rs2241057 in the CYP26B1 gene for patients with Ulcerative colitis and healthy controls, displayed for sub phenotypes and clinical features.(DOCX)Click here for additional data file.


Aurora-A V57I  polymorphism and cancer susceptibility has been widely studied. However, the results are inconsistent.The association between Aurora-A V57I polymorphism and the decreased risk of overall cancer in two genetic models: AA vs. GA+GG and AA vs. GG. In a stratified analysis by cancer type, significant association between Aurora-A V57I polymorphism and the decreased risk of breast cancer was identified in one genetic model: AA vs. GG. In a stratified analysis by ethnicity, in three genetic models, significant decreased cancer risk was observed among Caucasians  instead of Asians. Furthermore, a stratified analysis by ethnicity in breast cancer subgroup, five genetic models , significant decreased cancer risk was observed among Caucasians, but not among Asians. A slight publication bias was observed in our meta-analysis, thus nonparametric “trim-and-fill” method was utilized to detect the stability of our results. The adjusted odds ratios and confidence intervals showed that Aurora-A V57I polymorphism might be a protective factor for cancer risk, suggesting the reliability of our findings.To obtain a more precise evaluation of the relationship, we performed a meta-analysis of 14 case-control studies involving a total of 11,245 cancer cases and 16,024 controls. Our results demonstrated that there was a borderline evidence of an association between the Aurora-A V57I polymorphism may be a protective factor for cancer risk.In summary, this meta-analysis suggests that  Aurora-A coding region. F31I  is located on exon 3 of Aurora-A, encodes a phenylalanine → isoleucine substitution at amino acid residue 31 Aurora-A, encodes a valine → isoleucine at amino acid residue 57 Aurora-A V57I polymorphism was widely investigated for the association between the SNP and cancer risk. A number of investigations indicated that this SNP was a low protective polymorphism in carcinogenesis, particularly in breast cancer Aurora-A V57I polymorphism with cancer risk. Thus, we carried out a meta-analysis on overall eligible publications, to confirm whether the V57I polymorphism of Aurora-A was associated with cancer risk.Aurora-A protein, also known as STK15/BTAK, belong to the Aurora family of cell cycle-regulating serine/threonine kinase. Aurora-A plays a pivotal role of mitotic centrosome separation, maturation and spindle formation and stability Table S1. PRISMA checklist) The meta-analysis is reported on the basis of the Preferred Reporting Items for Meta-analyses (PRISMA) guideline  and CNKI  databases  using combinations of the following terms “Aurora-A”, “BTAK”, “stk15”, “AIKI”, “rs1047972”, “polymorphism”, “SNP”, “mutation”, “locus”, “cancer”, “carcinoma”, “tumor”, “neoplasm” and “malignance”. Additionally, in searching, the language of overall studies was restricted in English or Chinese. The references of retrieved publication, published reviews, letters and comments were scanned for additional relevant studies.Aurora-A V57I polymorphism and cancer risk; 2) case-control or cohort study design; 3) frequencies of genotypes or alleles in case groups and control groups could be exacted from the articles; 4) genotype distributions of controls passed Hardy-Weinberg equilibrium (HWE) test; 5) all cases were diagnosed by pathological examination.For inclusion in this meta-analysis, all eligible articles had to meet the major criteria: 1) focusing on the association of Exclusion criteria were: 1) overlapping data; 2) not case-control studies; 3) only relevant to oncotherapy; 4) review, meta-analysis or letter.In a standardized form, information from all original publications was extracted independently by two reviewers (W. Tang and H. Qiu). In case of disagreements, differences were adjudicated after elaborate discussion among all reviewers. For every eligible article, the following items were extracted: first author's name, cancer type, publication year, country, ethnicity of study subjects, sample size , genotype method, allele and genotype frequencies and evidence of HWE in controls.http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) was used to provide evidences of the HWE in the controls. STATA Version 12.0 software was used to calculate the crude odds ratio (OR) with the corresponding 95% confidence intervals (95% CI) to evaluate the strength of association between Aurora-A V57I polymorphism and cancer risk. Z test and P value (two-tailed) were used to evaluate the significance of the pooled OR, and if P<0.05, statistically significance was confirmed. In current meta-analysis, a Chi-square based I2 test was applied to assess the potential heterogeneity among eligible publications, with I2 value less than 25% indicating low heterogeneity; 25% to 50% indicating moderate heterogeneity; and greater than 50% indicating high heterogeneity 2>50% or P<0.10, the heterogeneity in publications was considered significant and the random-effects model (the DerSimonian-Laird method) was conducted for meta-analysis P<0.1 for the interpretation of Egger's test. Sensitivity analyses were conducted to evaluate the stability of the results. Nonparametric “trim-and-fill” method was also utilized to determine the stability of our results. All the statistical manipulations were implemented by using STATA 12.0 statistical software, and all the P values were two sided.An internet-based HWE test . After this step, 12 papers were identified for data extraction and assessment. After a manual search of the bibliography lists from retrieved, as a result, another two articles were recruited (Aurora-A V57I (rs1047972) polymorphism and cancer risk were recruited in this meta-analysis. Among 14 studies Aurora-A V57I genotype and allele frequencies among cancer cases and controls were presented in Relevant publications were retrieved from databases . As shown in ecruited Figure 1Aurora-A V57I polymorphism and decreased risk of cancer in two genetic models: AA vs. GA+GG  and AA vs. GG    in Caucasians, but not in Asians  in Caucasians, but not in Asians  Table 3.P = 0.511, Egger's test P = 0.113; AA vs. GG: Begg's test P = 0.443, Egger's test P = 0.083; GA vs. GG: Begg's test P = 0.324, Egger's test P = 0.271; AA vs. GA: Begg's test P = 0.324, Egger's test P = 0.190; dominant model: Begg's test P = 0.584, Egger's test P = 0.170; recessive model: Begg's test P = 0.511, Egger's test P = 0.103).Begg's funnel plot and Egger's test were carried out to measure publication bias. The shape of funnel plot revealed the evidence of funnel plot symmetry Figure 3Aurora-A V57I polymorphism may be a protective factor for cancer risk   polymorphism with cancer risk. However, the results were conflicting. Thus, we conducted a comprehensive meta-analysis involving published data, to assess the strength of association between Aurora-A V57I polymorphism and cancer risk. In current meta-analysis, a total of independent 14 case-control studies in 12 publications including 11,245 cases and 16,024 controls were recruited, and then identified the association of Aurora-A V57I polymorphism with cancer risk. Our results demonstrated a borderline association between the Aurora-A V57I polymorphism and decreased risk of cancer in two genetic models: AA vs. GA+GG and AA vs. GG. In a stratified analysis by cancer type, our results indicated that Aurora-A V57I polymorphism was associated with a significantly decreased risk of breast cancer in AA vs. GG genetic model. In a stratified analysis by ethnicity, a protective effect of Aurora-A V57I polymorphism was observed in Caucasians, but not in Asians. In addition, in a stratified analysis by ethnicity in breast cancer subgroup, five genetic models significant decreased cancer risk were observed among Caucasians , but not among Asians.In decade, several molecular epidemiological studies have been performed to assess the association of Aurora-A F31I polymorphism, converts the activity of the Aurora-A box-1, leading to obstruction of p53 binding and reducing the degradation of Aurora-A Aurora-A V57I polymorphism, which results in a valine  to isoleucin (Ile) substitution at codon 57, is associated with subdued celluar transformation and increased chromosomal stability. The Aurora-A V57I polymorphism in Aurora-A box-2 does not regulate Aurora-A degradation, but may affect the secondary structure and function of Aurora-A F31I polymorphism Aurora-A decreased the risk of cancer, perhaps by modifying the function of Aurora-A F31I polymorphism and altering the secondary structure of protein.It has been observed that the Aurora-A V57I polymorphism. The results indicated that Aurora-A V57I polymorphism was associated with a decreased risk of breast cancer, but not of ovarian cancer, lung cancer and other cancers. However, our results should be interpreted with very caution. Only five case-control studies for breast cancer, three for ovarian cancer and two for lung cancer were included in this meta-analysis, which might reduce statistical power to get a reliable result. Future, more large-scale studies should be conducted to confirm or refute these results. Because ethnicity could also affect the results of meta-analysis, we performed a stratified analysis by race for Aurora-A V57I polymorphism. Our results demonstrated the Aurora-A V57I polymorphism was a protective factor in Caucasians but not in Asians. In current meta-analysis, only three investigations of Asians were obtained. The outcomes might be due to fluke because the limited number of eligible studies and sample sizes might lead to deficient statistical power to measure a minor effect. Thus, our results should also be interpreted with very caution. In the future, further studies with large sample sizes regarding Asians should be carried out to identify the possible effects of Aurora-A V57I polymorphism ethnic variations on cancer risk.Since cancer origins might affect the outcomes from meta-analysis, we performed a stratified analysis by cancer type for Aurora-A V57I polymorphism may be a protective factor for cancer risk, suggesting the reliability of our findings.Two significant issues, heterogeneity and publication bias, should be addressed. A slight publication bias and relatively low heterogeneity were observed in our meta-analysis. As subgroup analyses were performed according to ethnicity and cancer type, Asian populations, lung cancer and breast cancer subgroups contributed the major source of heterogeneity. Since publication bias was observed, nonparametric “trim-and-fill” method was utilized to detect the stability of our results. The adjusted ORs and CIs show that Aurora-A V57I and F31I polymorphism locate on the same Aurora-A exon and significant linkage disequilibrium was found between the two coding SNPs, given the V57I polymorphism might play an important role in the process of carcinogenesis though altering the secondary structure and function of Aurora-A F31I polymorphism, thus, this important SNP, Aurora-A F31I, should not be ignored. Sixth, in this meta-analysis, some cancers only had one study included; therefore, any type of cancer investigated by less than two case-control studies was combined into “other cancers”, which might lead to heterogeneity in this subgroup.Although our results were stable and suggestive, there were several limitations in this study which should be acknowledged. First, only 14 eligible case-control studies in 12 publications were recruited in this meta-analysis; therefore, the results might be a fluke, as the limited number and sample sizes of eligible studies could lead to deficient statistical power to detect a real influence. Second, all eligible case-control studies were from Asians and Caucasians; thus, our results only proper for these two populations. Third, a slight publication bias was observed in our meta-analysis. Only twelve eligible publications were recruited in current meta-analysis, so some unpublished studies were unavoidably missed, which may result in bias. Fourth, high heterogeneity existed in some subgroups. This could be due to other differences between publications, such as age, gender, cancer type, ethnicity, smoking, drinking, other lifestyle factors, environmental risk factors and unscreened controls as well. For limited number of eligible studies or lack of sufficient information in a uniform criterion in investigations, these factors were not considered. Fifth, the Aurora-A V57I polymorphism may be a protective factor for cancer risk, especially, in Caucasians and breast cancer. As only 14 studies were recruited in this meta-analysis and current evidence was limited, in the future, more large-scale studies with an adequate methodological quality and properly controlling should be carried out, to confirm or refute the relationship between Aurora-A V57I polymorphism and the risk of cancer.In conclusion, this meta-analysis indicates that the Checklist S1PRISMA checklist, Checklist of items to include when reporting a systematic review or meta-analysis (diagnostic review consisting of cohort studies).(DOCX)Click here for additional data file.


Studies of twin pairs discordant for autoimmune conditions provide a unique opportunity to explore contributing factors triggered by complex gene-environment interactions.In this cross-sectional study, thirty-one monozygotic or dizygotic twin pairs discordant for myositis or systemic lupus erythematosus (SLE), along with matched healthy controls were evaluated for antibodies against a panel of 21 autoantigens.P = 0.046). Lastly, clinical analysis of both the affected monozygotic and dizygotic twins revealed that the autoantibody seropositive affected twins had a greater global disease activity score compared to seronegative affected twins (P = 0.019).Autoantibody profiling revealed that 42% of the affected twins showed significant seropositivity against autoantigens in the panel. In many of these affected twins, but none of healthy controls, there were high levels of autoantibodies detected against two or more autoantigens commonly seen in systemic autoimmune diseases including Ro52, Ro60, RNP-70 K and/or RNP-A. In contrast, only 10% (3/31) of the unaffected twins showed seropositivity and these immunoreactivities were against single autoantigens not seen in systemic autoimmune diseases. While no significant differences in autoantibodies were detected between the affected or unaffected twins against thyroid peroxidase, transglutaminase and several cytokines, 23% of the affected twins with myositis showed autoantibodies against the gastric ATPase. Analysis of the monozygotic twins separately also revealed a higher frequencies of autoantibodies in the affected twins compared to the unaffected twins (The findings of significantly more autoantibodies in the affected twins with myositis and SLE compared to the unaffected twins are consistent with potential non-genetic factors playing a role in autoantibody production and pathogenesis of these autoimmune disorders. Factors involved in the development of various autoimmune diseases including type I diabetes T1D), rheumatoid arthritis, myositis, and systemic lupus erythematosus (SLE) remain poorly defined. Studies of monozygotic twins with autoimmune conditions including multiple sclerosis, T1D, rheumatoid arthritis and SLE show a concordance rate of 20-70%, suggesting that the genetic makeup alone cannot completely explain the pathogenesis of autoimmunityD, rheuma-5. SpeciSystemic autoimmune diseases including systemic sclerosis, rheumatoid arthritis, SLE and idiopathic inflammatory myopathies (IIM) or myositis, are a group of immune disorders characterized by immune activation, autoantibody production and tissue destruction involving multiple organs. Although the target tissues and symptoms are markedly different among these diseases, a common molecular feature is the increased expression of interferon-regulated genes. For exaAutoantibodies are another common feature of these diseases, which have important clinical and prognostic utility. A numbeHowever, despite the widespread applications of autoantibody testing, only a limited number of studies have utilized autoantibody profiling to study cohorts of twins to dissect genetic and environmental factors that may contribute to systemic autoimmune disorders. In one small study of seven twin pairs discordant for SLE, 50% of the affected twins were found to have anti-DNA antibodies. Other sRecently, we have employed a liquid phase immunoassay, luciferase immunoprecipitation systems (LIPS), which utilizes light-emitting recombinant antigens to efficiently detect antibodies against linear and conformational epitopes associated with a variety of human autoantigens and infectious agents. Due to Informed written consent was obtained from all subjects in accordance with the human experimentation guidelines of the Department of Health and Human Services under multiple IRB-approved protocols, and the studies were conducted according to the principles expressed in the Declaration of Helsinki. Under IRB approved protocols at the National Institute of Environmental Health Sciences, NIH , 31 same-gendered twin pairs discordant for two different systemic autoimmune conditions (22 myositis and 9 SLE) were studied as part of the Twin-Sib study,30. FromRenilla luciferase fusion protein constructs were generated for detecting antibodies against additional known autoantigens essentially as described using the pREN2 vector[LIPS tests for many of the autoantigens used in this study have been previously described and included targets for systemic autoimmune diseases , Jo-1, RNP-A, and RNP-70 K), tissue autoantigens (thyroid peroxidase/TPO), transglutaminase/TGM2), the gastric ATPase and cytokines ,24,28. O2 vector. These n2, 1% Triton X-100 and 0.001% bromophenol red). For LIPS analysis, 40 μl of buffer A, 10 μl of diluted sera from the master plate , and 1 × 107 light units (LU) of Renilla luciferase-antigen Cos1 cell extract were added to a final volume of 100 μl) to each well of a standard polypropylene plate. After incubation for 1 hour at room temperature on a rotary shaker, the 100 μl antigen-antibody reaction mixture was transferred to a 96-well filter plate containing 5 μl of a 30% suspension of protein A/G beads and further incubated with shaking. For detecting anti-TGM2 IgA autoantibodies, goat anti-human IgA-agarose conjugated beads (Sigma) were substituted for protein A/G beads. After 60 minutes of incubation, the filter plates containing the bead-immobilized antibody-antigen complexes were washed using a BioMek robotic workstation with a vacuum manifold. The LU of the filter plates were then measured in a Berthold LB 960 Centro microplate luminometer  using coelenterazine substrate mix . Additional positive control sera and testing was also used for validating the diagnostic potential of some of the antigens. All data represent raw antibody levels without subtracting the buffer blanks. Based on known cut-offs for most of the autoantigens, seropositivity status was determined before the codes were broken. For the new autoantigens generated for this study, cut-off values were calculated based on the mean plus 3 standard deviations of the healthy controls.LIPS testing was performed as described in a detailed protocol and video. BrieflyP values were not corrected for multiple comparisons and P values less than 0.05 was deemed as statistically significant. The non-parametric Mann-Whitney U statistical test was used for comparison of antibody levels in the different groups. For comparing the seroprevalence in the different groups, contingency tables were generated and analyzed using the Fischer’s exact test for statistical significance.The antibody levels in the cohort were analyzed using the GraphPad Prism software . Since this study was exploratory, A heat map was employed for visualization of the spectrum and intensity of autoantibody responses in the individual seropositive twin pairs. For construction of the heatmap, the corresponding twin without autoantibodies was used as a reference group to determine the fold increase compared to the seropositive twin and was color-coded according to the key.A cohort of 31 disease-discordant twin pairs and 31 matched, healthy controls was utilized to study the prevalence of autoantibodies against a panel of defined human autoantigens. The clinical characteristics of the affected twins are described in Table U test; P = 0.0495).In total, 21 autoantigens were tested by LIPS . While 11 of the affected twins with SLE or myositis demonstrated seropositivity, none of the healthy twins (0/31) had autoantibodies against these targets. Evaluation by the Fischer’s exact test revealed that only the affected (n = 12) and not the unaffected twins (n = 0) had autoantibodies against the systemic autoantigen targets, and this difference was highly significant (P < 0.0001). Similarly, as expected, the affected twins had significantly higher frequencies of autoantibodies against these systemic autoantigen targets compared to the unrelated, healthy controls (P < 0.0001). Thus, our findings strongly suggest that autoantibodies against these targets are largely found in the affected twins and not in the unaffected twins or healthy unrelated controls.The frequency of autoantibody responses was also calculated for the eight autoantigens  seen in systemic autoimmune diseases that showed seropositivity in the cohort. From this analysis, 32% (7/22) of the twins with myositis and 44% (4/9) of the twins with SLE demonstrated seropositivity . Additionally, there were a total of 20 seropositive responses in the 7 affected monozygotic twins, but only one autoantibody response was found in one unaffected, monozygotic twin. In myositis, three of the 22 cases were from adults, but only the one subject with polymyositis showed significant autoantibodies. Lastly, inspection of the five ATPase seropositive DM patients revealed that they were all co-positive for Ro60, as well as one additional autoantigen, suggesting that the anti-gastric autoantibodies in myositis reflected existing high levels of auto-reactivity . Similarly, the global disease damage score was also significantly increased in the autoantibody positive patients (P = 0.047).From testing the 21 autoantigen panel, 15 autoantigens showed significant immunoreactivity with at least one of the unaffected or affected twins Table . As showWhile autoimmune diseases have high morbidity and mortality, little is known about the cause of most autoimmune disorders. Here auP = 0.046) suggesting that the production of autoantibodies likely involves more than genetic risk factors requiring additional epigenetic or environmental factors for inducing disease[Although three of the unaffected twins had autoantibody responses, the autoantibody levels were low and directed against three autoantigens, TPO, TGM2 and IFN-γ, which are not typically associated with systemic autoimmunity. Autoantibodies against TPO and TGM2 are also common in the general population, while the low levels of autoantibodies against IFN-γ in one unaffected twin was unlikely to have neutralizing cytokine activity. Moreover, the corresponding affected twins in each case were not found to harbor these three autoantibodies. The presence of autoantibodies in three unaffected twins may reflect normal, acute inflammatory conditions, such as those seen in response to infection, or possibly other immune-mediated abnormalities observed among relatives of patients with autoimmune conditions. A key finding in our study was that 31.8% of the monozygotic affected twins were autoantibody seropositive vs. only 4.5% of the unaffected, monozygotic twins (Based on our previous study, approximately 90% of SLE patients demonstrated autoantibodies against five of the candidate autoantigens. SurprisOne novel finding of our study was the detection of autoantibodies directed against the gastric ATPase in several myositis patients that were also positive for other autoantigens. Previous studies have identified autoantibodies against the gastric ATPase in other autoimmune conditions including autoimmune gastritis, SjögrenIn contrast to the study by Reichlin et al., we founIn our cohort of relatively young twins discordant for myositis or SLE, our results show dramatic differences in autoantibody profiles between affected and unaffected twins. The clear difference between the autoantibody levels of the seropositive patients and the subset of corresponding unaffected twins supports the idea that the developmental onset of autoantibody production is controlled by more than genetic risk factors. Based on the observed induction of autoantibodies before disease onset, more deANA: Anti-nuclear antibodies; DM: Dermatomyositis; FDR: First-degree relatives; IFN: Interferon; IIM: Idiopathic inflammatory myopathies; LIPS: Luciferase Immunoprecipitation Systems; PM: Polymyositis; SLE: Systemic Lupus Erythematosus; Sm: Smith autoantigen; TGM2: Transglutaminase 2; TPO: Thyroid peroxidase; PO: Ribosomal protein PO.The authors declare that they have no competing interests.Contribution: LG contributed to study design, sample preparation, data analyses, and manuscript preparation. TPO’H contributed to study design, sample preparation, and manuscript editing. AG contributed to LIPS test, data analysis and manuscript editing. LGR contributed to patient recruitment, clinical assessments, and manuscript editing. FWM contributed to study design, patient recruitment, clinical assessments, and manuscript editing. P.D.B contributed to study design, LIPS test, data analyses, and manuscript drafting and editing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/15/67/prepub