Campylobacter. Because horizontal transmission from the environment is thought to be the most likely source of Campylobacter to broilers, we aimed to identify broiler house characteristics and management practices associated with flock colonization. Between May 2001 and September 2004, pooled caecal samples were obtained from 1,425 flocks at slaughter and cultured for Campylobacter. Due to the strong seasonal variation in flock prevalence, analyses were restricted to a subset of 792 flocks raised during the four summer seasons. Logistic regression models with a farm random effect were used to analyse the association between flock Campylobacter status and house-level risk factors. A two-stage process was carried out. Variables were initially screened within major subsets: ventilation; roof and floor drainage; building quality, materials and repair; house structure; pest proofing; biosecurity; sanitation; and house size. Variables with p ≤ 0.15 were then offered to a comprehensive model. Multivariable analyses were used in both the screening stage (i.e. within each subset) and in the comprehensive model.The concurrent rise in consumption of fresh chicken meat and human campylobacteriosis in the late 1990's in Iceland led to a longitudinal study of the poultry industry to identify the means to decrease the frequency of broiler flock colonization with Campylobacter colonization was predicted to increase when the flock was raised in a house with vertical (OR = 2.7), or vertical and horizontal (OR = 3.2) ventilation shafts, when the producer's boots were cleaned and disinfected prior to entering the broiler house (OR = 2.2), and when the house was cleaned with geothermal water (OR = 3.3).217 out of 792 flocks (27.4%) tested positive. Four significant risk factors were identified. Campylobacter. Although it remains unclear how geothermal water increases risk, further research is warranted to determine if it is a surrogate for environmental pressures or the microclimate of the farm and surrounding region.The increased risk associated with vertical ventilation shafts might be related to the height of the vents and the potential for vectors such as flies to gain access to the house, or, increased difficulty in accessing the vents for proper cleaning and disinfection. For newly constructed houses, horizontal ventilation systems could be considered. Boot dipping procedures should be examined on farms experiencing a high prevalence of  Campylobacter spp. remain one of the most frequent bacterial causes of foodborne gastroenteritis world-wide [Campylobacter to humans [Campylobacter spp. varies, ranging from 5% of flocks to more than 90% [Campylobacter in broiler flocks and production of birds free from colonization at slaughter, is essential for preventing human cases [rld-wide . Poultryo humans -7. The pthan 90% . Once a than 90% . Due to an cases -17.Campylobacter to broiler flocks is the environment . Both Campylobacter-positive and -negative flocks can be present in different houses on the same farm during the same growing period [Campylobacter from entering a broiler house. Thus, it might be hypothesized that certain characteristics of a broiler house, or management practices at the house-level, might influence the likelihood that a flock will be exposed to the bacteria. House-level factors associated with an increased risk of colonization include: concrete floors (compared to wood floors) [Campylobacter status and the attributes and management practices of broiler houses in Iceland has not been studied.Many researchers ,15,18-28g period ,11,12, i floors) ,16,29-31 floors) ; a stati floors) ; two or  floors) ; non-cem floors) ; and poo floors) . In Icel floors) ; howeverCampylobacter, thereby reducing the burden of foodborne illness associated with poultry consumption. Our objective in this study was to identify house-level risk factors associated with the colonization of broiler flocks with Campylobacter spp. in Iceland.The strong association between the increased incidence of human campylobacteriosis and increased consumption of fresh chicken meat in Iceland in the late 1990's prompted a longitudinal study of the poultry industry . The ultCampylobacter. The 792 flocks originated from 83 houses on 33 farms. The median number of flocks per house was 10 . Ten houses did not have any positive flocks; the number of flocks raised in these houses ranged from 1 to 15, with the majority having ≥ 9 flocks per house. The distribution of the proportion of positive flocks per house is shown in Figure There were 792 flocks raised in the summer seasons, and of these, 217 (27.4%) tested positive for Of the 217 positive flocks, 157 (72.4%) were slaughtered in 1 catch lot , 46 (21.2%) were slaughtered in 2 catch lots , and 14 (6.5%) were slaughtered in 3 or 4 catch lots . For flocks with more than one catch lot, the number of days between the first and last catch lot was relatively short , and 82.2% of the flocks with multiple catch lots were positive in samples collected from the first catch lot. On the basis of catch lot sampling, out of 291 positive catch lots, 266 (91.4%) were positive in all 4 pooled samples, 2 (0.7%) were positive in 3 pooled samples, 6 (2.1%) were positive in 2 pooled samples, and 17 (5.8%) were positive in only 1 pooled sample.-15). Based on these observations, a random effect at the farm level was included in all models to control for clustering.The intra-class correlation coefficient (ICC) of a null model with only a house random effect was 0.10 (p < 0.001); the ICC of a null model with only a farm random effect was 0.21 (p < 0.001). When both random effects were included together in a null model, the variance of the farm random effect (0.6) was substantially higher than the variance of the house random effect , or vertical and horizontal (OR = 3.2) ventilation shafts, when the producer's boots were cleaned and disinfected prior to entering the broiler house (OR = 2.2), and when the house was cleaned with geothermal water (OR = 3.3)  predictors. The risk of tal OR = .2 ventil-7, p = 1.000) of the final model was extremely small, indicating that there was very little clustering at the farm-level after accounting for the variables in the model. Thus, performing diagnostics on the model without the random effect was deemed to be acceptable. The final model included 11 covariate patterns. The Pearson χ2 goodness-of-fit test was not significant (p = 0.12) indicating that we could not reject the null hypothesis that the model fit the data. There was one covariate pattern that had a standardized Pearson residual of 2.96 . Ninety out of 212 flocks (42%) with this pattern were positive for Campylobacter, and the predicted probability of a positive flock was 39%. Flocks with this covariate pattern were raised in houses with vertical ventilation, the producer's boots were always cleaned and disinfected prior to entering the house, and geothermal water was used to clean the houses. Although this covariate pattern had the largest leverage (0.87) and delta-beta (58.1) values, it also had the highest number of observations (n = 212), thus, its moderate influence on the model was not of great concern.To assess the effect of performing model diagnostics on the final model without a random effect, we compared the parameter estimates of our final model to the same model without a random effect at the farm level, and found that the estimates remained stable. The ICC . The observed percentage of positive flocks with this pattern was 38% (51 out of 133), and the predicted probability was 42%. Flocks with this covariate pattern were raised in houses with both vertical and horizontal vents, boots were always cleaned and disinfected prior to entering the house, and geothermal water was used to clean the houses. This covariate pattern had the second highest leverage value (0.79), delta-beta value (17.1), and number of observations (n = 133), thus, its influence on the model was of little concern.Campylobacter spp. in Iceland, out of a possible 30 explanatory variables. The higher ICC of a null model with a random effect at the farm level, compared to a null model with a random effect at the house level, suggests that there was more variation in flock Campylobacter status between farms than within farms. Thus, it was not unexpected that only a few house-level predictors remained statistically significant in the final model. In addition, our approach of screening variables within subsets enabled us to look at potentially complex relationships between predictors of a similar type, and identify those that were most strongly associated with flock status. For example, in some subsets (e.g. sanitation), including more than one predictor in the model led to unstable parameter estimates with high standard errors, therefore, only one predictor from the subset could be offered to the comprehensive model. Within other subsets, there was strong collinearity between variables, or between one variable and one category of another variable. For example, all six houses  in which the roof and floor drains merged, also had roof drains that opened into a septic tank with overflow into an underground bed . In these subsets, only one of the two variables could be included in the analysis; the predictor resulting in the model with a smaller Akaike's Information Criteria (AIC) value was chosen. Additionally, there were several variables in which the responses were extremely one-sided; almost all producers cleaned the ventilation system after every flock and changed boots before entering the house, and all but one broiler house in Iceland have concrete floors (the floor of the upper level of one house included wood elements). These variables were eliminated from the analysis because it was unlikely that there would be enough power to detect such small differences.Our study has identified four house-level risk factors associated with the colonization of broiler flocks with Misclassification could have been a potential source of bias in our study. For some variables, it was necessary to combine categories if there were only a small number of observations in one or more categories. However, we expect this bias was small because we grouped only those categories that were biologically-related, and only when a Wald's test indicated that the categories were not statistically different from each other. In addition, eliminating predictors due to problems with collinearity or unstable parameter estimates might have affected our final results. However, we found that predictors that were strongly associated with flock status in the subset analyses, were also statistically significant in the final model. Thus, it is likely that our screening process was effective in identifying the most important of the house-level predictors.Campylobacter. In a previous analysis of flock-level factors [Campylobacter, after controlling for flock size. Most of the positive catch lots in our study were positive in all four pooled caecal samples, inferring high sensitivity of the methodology for the sample type. On a flock basis, 84% of the positive flocks yielded Campylobacter in all samples collected, and of the positive flocks with more than one catch lot, a high proportion were positive on the first catch lot. Several standard management practices in Iceland might have contributed to these findings. Beginning in 1999, before the start of the study period, the Icelandic poultry industry adopted a high standard of cleaning, chemical disinfection and drying of live haul crates and trucks, under the assumption that bringing crates covered with faecal material into broiler houses for partial depopulation was an important source of contamination to the remainder of the flock. Unlike other countries, Iceland does not have commercial catching crews that travel from farm to farm. The workers on each farm catch their own birds and follow their own biosecurity rules. In addition, the reason for collecting and slaughtering a flock in multiple catch lots differs in Iceland compared to other countries. In Iceland, the practice is based more on slaughter line speed capacity in the abattoirs; only the largest flocks  are slaughtered in three or four catch lots, typically over three or four consecutive days. For flocks slaughtered in more than one catch lot, the average interval between catch lots is quite short. The practice of thinning  is rare in Iceland, and is typically done only during the Christmas season. During the winter, the prevalence of Campylobacter in broiler flocks is very low (see Methods). Therefore, because of the relatively short time interval between shipping catch lots, the low within-flock prevalence used in our sample size calculation to detect early stages of colonization, the high sensitivity of the isolation method, and the high proportion of catch lots that were positive in all samples, we expect that there was little variation with respect to the risk of false negative classification between flocks slaughtered in single and multiple catch lots.For flocks with multiple catch lots, more pooled samples were obtained, and we considered whether this might have increased the likelihood that the flock would be positive for  factors , the numCampylobacter jejuni [Campylobacter prevalence has been shown to be higher on farms with frequent sightings of wild birds than on farms with few wild birds [Campylobacter to broiler flocks, we classified the ventilation system on each broiler house according to its type , and integrity (open or closed). An underlying assumption of our classification was that wild birds might be more likely to perch on vertical ventilation shafts, and, if those vents were open, the flock might be more likely to be exposed to the bacteria, either directly (e.g. wild birds entering the house or defecating into the vents), or indirectly . From the subset analysis, we found that after controlling for ventilation type, the integrity of the ventilation system was not a significant risk factor for flock colonization . However, the risk of colonization was predicted to increase when the flock was raised in a house with vertical, or vertical and horizontal ventilation shafts compared to horizontal vents. There are a few possible reasons for these findings. First, our classification of ventilation type might not have been refined enough . Although researchers in Sweden [Campylobacter occurrence and air outlets  or inlets , the authors stated that their conclusions might have been uncertain because confounding  and clustering were not accounted for in the analysis. Secondly, there might be some mechanism related to the type of ventilation system other than wild birds that contribute to flock colonization. Our finding of a protective effect of horizontal shafts might be related to the thoroughness of house sanitation, as it has been suggested that horizontal fans are more accessible for proper cleaning and disinfection [Campylobacter such as flies. Researchers in Denmark [Campylobacter, and that per volume of ventilation air, 4.5 times the number of flies entered the house through a roof inlet than through wall inlets. Considering our findings, and those of Hald et al. (2004), further investigation in this area is warranted.Wild birds captured on broiler farm premises frequently carry r jejuni ,28,36-39ld birds . A previld birds  has founn Sweden  did not nfection . A brief Denmark  have shoCampylobacter colonization was predicted to increase when the flock was raised in a house in which the producer's boots were cleaned and disinfected prior to entering the house. Using univariable analyses, several researchers [Campylobacter-positive flocks with a decreasing hygiene score (including the disinfection of boots), although the hygiene score itself was not statistically significant. Campylobacter jejuni has been isolated from farmer's boots [The risk of earchers -31,33 haearchers  have not's boots ,19,22,39's boots . Several's boots ,16,21 ha's boots  noted thCampylobacter colonization was higher when the flock was raised in a house cleaned with geothermal (high temperature) water. The isolation of Campylobacter from surface water [Campylobacter to broiler flocks. Although not all geothermal wells were on-farm , potentially, farm-based geothermal wells could have warm surface water pools, which might serve as Campylobacter reservoirs for birds, flies and other insects, and for the broiler flocks. However, several researchers [Campylobacter spp. from various water sources were highest when the water temperature was between 2°C and 10°C, and lowest when the temperature exceeded 15°C. Thus, the reason for the positive association between Campylobacter status and the use of geothermal water for cleaning remains unclear. In Norway, the prevalence of Campylobacter in three surface water sources (lakes and rivers) was strongly associated with the number of indicator bacteria  in the water [Campylobacter . To our knowledge, this is the first time this risk factor has been identified in the literature, and it will be of interest to ascertain if this predictor is recognized in future studies and to determine the mechanism of its effect.Our study has shown that the risk of ce water  and puddce water  adjacentearchers -47 have he water . TherefoCampylobacter in broiler flocks in Iceland; vertical or vertical and horizontal ventilation shafts, cleaning and disinfecting boots prior to entering the broiler house, and the use of geothermal water for cleaning houses. We found that horizontal vents had a protective effect, therefore, extra care should be taken when cleaning vertical vents, and producers should ensure that the disinfectant used has an appropriate bactericidal effect for Campylobacter. Alternatively, the increased risk associated with vertical ventilation shafts might be related to the height of the vents and the potential for vectors such as flies to gain access to the house. This is an area that warrants further research, and further refinement in the classification of vertical and horizontal systems might be necessary for this purpose. For newly constructed houses, horizontal ventilation systems could be considered. Boot dipping procedures, including the frequency and thoroughness of dipping boots, the frequency of changing the dip, and the effectiveness of the dip against Campylobacter should be examined on farms experiencing a high prevalence of Campylobacter. Future studies are warranted to ascertain how geothermal water increases the risk of colonization, and to determine if this factor is a surrogate for micro-climatic conditions or agro-environmental pressures on the farm and surrounding region.Our study has identified four risk factors related to broiler house attributes and house-management practices, for the occurrence of The target population was commercial broiler chicken flocks raised in Iceland between May 2001 and September 2004. The study population included all broiler flocks produced by the three largest poultry companies in Iceland during the study period. In total, only 149 flocks  from three farms in the north of Iceland and a coastal island were excluded, due to their remote location and associated difficulty in collecting data and samples.Data on the characteristics of each farm and broiler house were gathered at the beginning of the study through a combination of phone interviews and site visits by the Veterinary Officer for Poultry Diseases of the Agricultural Agency of Iceland. Farms were also visited frequently during the study, and the recorded information was verified, including changes that occurred over time .A questionnaire was used to collect house-level epidemiological data; this was administered by one of two field technicians of the Veterinary Officer for Poultry Diseases in a face-to-face interview with the producer after each flock was shipped for slaughter. Major subsets of variables included: ventilation; roof and floor drainage; building quality, materials and repair; house structure; pest proofing practices; biosecurity measures; sanitation practices; and house size. To ensure consistency in responses, data collected at the previous visit were reviewed with the producer, and any changes that occurred from one flock to the next were recorded.Campylobacter is likely influenced by factors acting at more than one level of production , and other factors potentially relevant to the epidemiology of Campylobacter were recorded, it was our intent in this study to restrict the analysis to factors acting specifically at the broiler-house level. Understanding the complex relationships between a large number of house-level management practices and broiler house characteristics , and identifying the variable(s) from each major subset most strongly associated with flock Campylobacter status, was deemed necessary to direct interventions that might prevent Campylobacter from entering a broiler house (and thus prevent flock exposure). The group of factors chosen for this analysis were considered both sensible and comprehensive to satisfy the objectives of this study and were consistent with house-level factors reported in the literature.Although the colonization of broiler flocks with Campylobacter colonization or alleles with poor colonizing ability on the basis of a within-flock prevalence as low as 10%; four pooled samples would ensure 99% confidence of detecting at least one positive bird in a catch lot [Campylobacter isolation in Nordic countries, the Campy-Cefex direct plating method has a sensitivity and specificity of 97.8% and 97.6% (on a catch lot basis), and a sensitivity and specificity of 98.8% and 97.3% (on a pooled sample basis), respectively, for detecting Campylobacter spp. in broiler caecal samples at slaughter [Campylobacter spp., high sensitivity, and the ability to enumerate samples. Colonies were counted, and confirmed as Campylobacter spp. by microscopy and latex agglutination. A broiler flock was considered positive for Campylobacter if at least one of the pooled samples from any of the catch lots was positive on culture.Monthly reports summarizing records of flocks slaughtered each day were obtained from the processing plants. Flocks were collected and slaughtered in one to four catch lots depending on their size and on-farm management practices. At the processing plants, systematically selected caeca (including contents) were excised from 40 birds from each catch lot by the plant veterinarian and placed in sterile plastic bags to create four pooled samples containing ten caeca each. Samples were processed either the same day or after holding overnight at 4°C. The required sample size per flock was estimated to detect early stages of flock atch lot . Serial atch lot  and inculaughter . The CamCampylobacter colonization of broiler flocks has been well-described in the literature. Over the full 3 1/2 year study period, 227 out of 1,425 flocks (15.9%) tested positive for Campylobacter. Almost all of the positive flocks (217 out of 227) were raised during the summer season (hatch dates between March 15 and September 15 of each year of the study). As a result of the strong seasonal variation in flock prevalence, it was of interest to focus our analysis on flocks raised during this high risk summer period. Our definition of summer corresponds to the periods of restrictions imposed by the Icelandic government on when manure is allowed to be spread on fields and pasture (March 15 to October 31).A summertime seasonal pattern of A house-level variable was considered to be a physical characteristic of the broiler house, or a management practice that was carried out at the house-level. We had initially assumed that these variables would be relatively consistent for all flocks raised in the same house during the study period. However, producers occasionally instituted changes, such that not all flocks raised in the same house were exposed to the same attribute or subjected to the same management practice. As a result, we were unable to collapse the data to the house level.The data were analysed in two stages. Variables were initially screened (stage one) within logical subsets of risk factors Tables  and 2. AThe process of variable selection was similar for all models and stages. For categorical predictors with more than two levels, a likelihood ratio test was used to assess the contribution of the variable to the model; for continuous and dichotomous predictors, a Wald's test was used. In addition, the AIC was used to compare non-nested models. As each variable was removed from a model, its effect on the coefficients of the other variables in the model was assessed. If the coefficient of another significant variable changed by more than 30%, the variable was deemed to be a confounder and was forced into the model. Once a main effects model was chosen, 2-way interaction terms were introduced one at a time and evaluated for statistical significance using either a Wald's test or likelihood ratio test. All statistical analyses were performed using Stata software version 8 .Prior to modelling, frequency tables were used to scrutinize all categorical variables within each subset. Dichotomous predictors were excluded from the subset analysis if there were ≤ 10 observations in one category. For predictors with more than two levels, categories were combined if there were a small number of observations (≤ 15) in one or more categories, and/or to group biologically meaningful categories together.Campylobacter. This led to very high standard errors of the parameter estimates. In these situations, the predictor with the strongest univariable association with Campylobacter status was made available to the comprehensive model.In some cases, having two or more predictors in the same subset led to the occurrence of cells with very low frequencies of observations. For example, when a chemical cleaner was used to clean the house, but a bacterial disinfectant was not used, only three flocks were positive for b with adjustment for ties for categorical variables, and Pearson's correlation coefficient (r) for continuous variables). If one variable was collinear (τ or r ≥ 0.8) with another variable (or category of another variable) in the same subset, the predictor with the strongest univariable association was made available to the comprehensive model. If the models were not nested, the predictor resulting in the model with a smaller value of the AIC was made available.Within each subset, correlations between predictors were evaluated (Kendall's τContinuous predictors are summarized in Table Significant predictors from each subset Tables  and 2 we2 goodness-of-fit test to assess the overall fit of the model, with p ≤ 0.05 indicating a poor fit. Deviance and standardized Pearson residuals were calculated on the basis of one per covariate pattern; patterns with residuals less than or greater than 2.0 were examined. Potential influential observations were identified by examining large leverage and delta-beta [Due to the limitations associated with assessing residuals and other diagnostics in a random effects model in Stata, diagnostics were performed on the model without the random effect. We used the Pearson χlta-beta  values.MTG performed the statistical analysis and drafted the manuscript. WM, OB and SAM critically evaluated the analysis and revised the manuscript for intellectual content. JR was involved in the conception, design and coordination of the study, data collection and data quality checks, and revision of the manuscript for intellectual content. JRB was involved in the conception and design of the study, the design of the epidemiological database structure, building the data query for the statistical analysis, and revision of the manuscript for intellectual content. RL was involved in the conception, design and coordination of the study, data management and final data quality control, and revision of the manuscript for intellectual content. All authors read and approved the final manuscript.


This study was designed to investigate the association of the 894G>T polymorphism in the eNOS gene with risk of acute myocardial infarction (AMI), extent of coronary artery disease (CAD) on coronary angiography, and in-hospital mortality after AMI.We studied 1602 consecutive patients who were enrolled in the GEMIG study. The control group was comprised by 727 individuals, who were randomly selected from the general adult population.The prevalence of the Asp298 variant of eNOS was not found to be significantly and independently associated with risk of AMI , extent of CAD on angiography  and in-hospital mortality .In contrast to previous reports, homozygosity for the Asp298 variant of the 894G>T polymorphism in the eNOS gene was not found to be associated with risk of AMI, extent of CAD and in-hospital mortality after AMI The functional significance of this single nucleotide polymorphism remains an issue of controversy since homozygosity for the Asp298 variant has been related to reduced enzyme activity [A single base exchange  is a multicenter study designed to evaluate the genetic predisposition of AMI and prognosis after AMI in the Greek population. A total of 1602 consecutive patients, admitted in hospital with the diagnosis of AMI, who have been successfully genotyped for the 894G>T polymorphism were enrolled in the study. The control group consisted of 805 adults (aged > 30 years old) who were randomly selected from the city records. Blood samples for genetic analyses were obtained from 794 of the study participants. Thirty-two out of the 794 eligible subjects had clinical or electrocardiographic evidence of a possible MI and were not included in the control group. Out of the remaining 762 subjects, 727 were successfully genotyped for the 894G>T polymorphism. The study methodology, participating centers, study investigators and findings have been published in details elsewhere -20. The The Greek population is a relatively homogeneous Caucasian population in terms of ethnic and cultural background, which presents low ischemic heart disease mortality rates and low incidence of CAD despite the relatively high prevalence of major coronary risk factors. The aforementioned characteristics have been verified in large scale epidemiological studies in the general population  and in pMboI restriction endonuclease digestion for 16 hours at 37°C. Non-denaturing, 3% agarose gel electrophoresis, was used to identify a single PCR product of 206 bp. Cleavage of the product into 119 bp and 87 bp fragments occurred in the presence of a T at nucleotide 894. The latter corresponds to Asp298, while no restriction digestion products were obtained in the absence of T at the same residue.In order to investigate the 894G>T polymorphism of eNOS gene, located in exon 7, oligonucleotide primers for polymerase chain reaction (PCR) were designed using the published sequence of the human eNOS (NOS 3) gene (Genbank/EMBL L10693–L10709) . A codinStatistical analyses were performed with SPSS software . A P value on a 2-sided test of 0.05 for group comparisons and 0.10 for interaction tests was considered statistically significant. Chi-square tests were used to compare genotype frequencies in different groups. Multivariate logistic regression analysis, with an allowance for age only, age and gender, or a group of the major cardiovascular risk factors , was used to explore the impact of Asp298 variant on risk of AMI. Chi-square analysis was used to test the assumption of Hardy-Weinberg equilibrium.2 = 0.20, P > 0.1) and controls .The main baseline characteristics of the patients with AMI (cases) and of the subjects from the general population (controls) are presented in Table The frequency of the Asp/Asp genotype was not found to differ significantly between cases and controls in relation to the major coronary risk factors . Furthermore, the average number of the aforementioned risk factors did not differ significantly in carriers versus non-carriers of the Asp/Asp genotype, in both cases  and controls .The frequency of the mutated genotype (Asp/Asp) did not differ significantly between cases and controls . Similarly, the Glu/Asp  and the Glu/Glu  genotypes did not differ significantly between the study groups in univariate analysis. In backward stepwise logistic regression analysis with age, gender, smoking status, diabetes mellitus, hypercholesterolemia, hypertension, obesity and family history of CAD included as covariates, the presence of the Asp/Asp genotype was not found to be independently associated with AMI  . Possession of this genotype was also not significantly associated with in-hospital mortality in multivariate analysis adjusted for age, gender, diabetes mellitus, hypertension, smoking status, obesity, anterior location of infarction, administration of thrombolysis, and echocardiographic left ventricular ejection fraction  . In multivariate analysis adjusted for age, gender, diabetes mellitus, smoking status, hypertension, hypercholesterolemia, and obesity, the Asp/Asp variant was not found to be independently associated with the number of diseased vessels on coronary angiography   and final phenotype (AMI) of interest.894→T polymorphism in the eNOS gene with risk of AMI which had been verified in a series of relatively small association studies.In conclusion, this relatively large scale study, devalues the hypothesized association of the GThe authors declare that they have no competing interests.GKA and EWH carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. DKG, SET and DCS carried out the immunoassays. SIZ and DJR participated in the sequence alignment, SGF, MNZ and EJG participated in the design of the study and performed the statistical analysis. ASM, PKT and CIS conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


We also quantified LOXL1 mRNA expression in the human lens capsule to examine the possible correlation between LOXL1 expression and XFG pathogenesis.We performed genetic association studies using a native Japanese population to examine the reproducibility of results of We performed a case-control study using 95 Japanese XFG patients and 190 controls. Real-time polymerase chain reaction (PCR) analysis was performed using lens capsules obtained during surgery.rs1048661 and the GG genotype in the SNP rs3825942 in exon 1 of LOXL1 were significantly associated with an increased risk of XFG under recessive models . Quantification of LOXL1 mRNA expression demonstrated no significant difference between XFG and senile cataract samples.The TT genotype in the single nucleotide polymorphism (SNP) LOXL1 SNP appear to be qualitative rather than quantitative, the amino acid substitution (R141L) caused by SNP rs1048661 is not a simple decisive factor for XFG due to the inverted allele frequency between Japanese XFG and Caucasian XFG patients. Further genetic and functional studies are essential for clarifying XFG pathogenesis.Although the functional effects of the  One of the nonsynonymous SNP (rs1048661) showed a very strong association with XFG. However, the risk allele was inverse compared to the Caucasian study. To gain further insight into the role of LOXL1 for XFG, it is important to compare the expression levels of LOXL1 mRNA in XFG eyes and non-glaucomatous eyes. We obtained anterior lens capsules during combined glaucoma-cataract surgery or during cataract surgery alone from XFG patients and non-glaucomatous senile cataract patients, respectively. We then performed a quantitative analysis of LOXL1 mRNA expression using these anterior lens capsules.Exfoliation glaucoma (XFG) is an age-related disorder associated with exfoliation syndrome (XFS), manifested by abnormal fibrillar deposits on the lens and iris epithelium . Recentl XFS/XFG . It was  XFS/XFG , therefoAll XFG patients were diagnosed by slit-lamp examination for the existence of exfoliation material on the anterior lens capsule with maximal dilation of the pupils and with glaucomatous optic neuropathy as well as visual field defect. Peripheral blood was obtained from 95 XFG patients 47–93 years of age (mean age: 75.7±8.1 years). The controls were 190 randomly-selected, population-based individuals 54–83 years of age (mean age: 65.0±6.8 years) with no glaucomatous changes or existence of exfoliation materials . All of rs1048661 and rs3825942) in the LOXL1 gene region according to the previous report [We genotyped two nonsynonymous single nucleotide polymorphisms  and 95% confidence intervals (95% CI) were also calculated.The frequencies of the genotypes were compared between XFG patients and controls in the recessive model. In this model, frequencies of the homozygous genotype for major alleles were compared using a 2x2 contingency table. Here, the association was evaluated using the χLOXL1 (Hs00173746_m1), and 18S rRNA from Applied Biosystems (Assay-on-Demand gene expression products). Real-time PCR analysis was performed on a 7500 real-time PCR system. The relative expression of LOXL1 mRNA in the anterior lens capsule was quantified by the standard curve method using 18S rRNA expression in the same cDNA as the control.Anterior capsules that were obtained during glaucoma/cataract or cataract surgery with written informed consent were immediately stored with RNAlater reagent  to protect the RNA. All procedures were approved by the ethics committee of Kyoto Prefectural University of Medicine. Total RNA was isolated with the Micro RNA extraction kit  from the anterior capsules, and then cDNA was prepared as described previously . The antrs1048661 TT genotype and the rs3825942 GG genotype in the first exon of LOXL1 were significantly associated with an increased risk of XFG under recessive models . The results obtained by the two methods were identical.We genotyped 95 XFG cases and 190 control subjects by direct sequencing methods. The rs1048661 and rs3825942 and found that those two SNPs are in a state of strong LD (Haploview [rs1048661 G/T and rs3825942 G/A), the TG haplotype showed an increased risk for XFG  in recessive models. We checked HWE among control subjects using this two-locus haplotype and found that the two-locus haplotype was concordant with HWE .First, we checked the state of linkage disequilibrium (LD) between trong LD . Next, wtrong LD  using HaLOXL1 mRNA in the lens epithelium obtained from XFG patients and senile cataract patients. (-test)cDNA was synthesized from total RNA isolated from the anterior lens capsules of patients undergoing cataract surgery. We analyzed both XFG (n=10) and senile cataract (n=10) specimens. LOXL1 SNP and XFG patients. The lysyl oxidase protein family has multiple functions including specific oxidative deamination of lysine residues and the cross-linking of elastin [LOXL1 knockout mice [LOXL1 is one of the causative genes for XFG.Similar to previous studies among Caucasian ,7,8, we  elastin . Phenotyout mice  showed tout mice . Therefors3825942 genotype, our results are consistent with those of Thorleifsson et al. [rs3825942 G is consistently higher among Caucasian and Japanese XFG patients so it is reasonable to conclude that rs3825942 A is a protective allele against the occurrence of XFG.For the rs1048661-“T” among the Japanese population and rs1048661-“G” among the Caucasian population. We double-checked these genotype results (rs3825942 and rs1048661) by both direct sequencing and the TaqMan genotyping assay because the genotype results of rs1048661 within control subjects were not concordant with HWE. The results obtained from two different methods were in complete agreement. Therefore non-concordance with HWE is not due to genotyping errors. We further checked the two-locus haplotype and found that the haplotype was concordant with HWE. Since the two-locus, rs1048661 and rs3825942, was in the state of linkage disequilibrium (D’=1.0), it was appropriate to check the HWE by haplotype.On the other hand, the risk allele for XFG occurrence is rs1048661 G/T genotype may have a protective role against XFG occurrence, or (3) there might be another causative polymorphism in a state of linkage disequilibrium with rs1048661 G/T. If the last possibility is the case, there might be a historical recombination between the SNP rs1048661 and the causative polymorphism.Inverted genotypes of XFG patients between the Japanese and Caucasian population suggests the following possibilities: (1) The 141st amino acid substitution (R141L) does not have a dominant role for the pathophysiology of XFG, (2) the heterozygote for the LOXL1 mRNA expression in anterior lens capsules and found that there was no significant difference between XFG and senile cataract controls. These results are different from the previous study by Thorleifsson et al. [LOXL1 mRNA expression with the TT genotype (the protective genotype against XFG in the Caucasian population) than those with the TG or GG genotype using adipose tissues. We hypothesize that the difference might be due to the mixed LOXL1 SNP genotype of our senile cataract surgery samples, which are not able to clarify genotype due to ethical procedure reasons. However, our LOXL1 mRNA expression analysis reflects a direct pathological site within the ocular tissue and better represents LOXL1 expression status in the affected eyes than that of adipose tissue [LOXL1 mRNA is not a direct pathogenetic cause of XFG.As a next step, we quantified relative n et al. , which se tissue . TherefoLOXL1 for XFG, we are now performing extensive SNP discovery around LOXL1 to find out other possible causative polymorphisms. In addition, functional analysis of LOXL1-fibulin5 protein interaction [LOXL1 and its mixture to gain further insight into our genetic association results showing that the rs1048661 G/T heterozygote is protective against XFG. It is also important to investigate the protective role of LOXL1 protein variants that possess the 153D amino acid, which is indicated by multiple genetic association studies including our own [To further clarify the pathophysiological role of eraction  using tweraction ,12, we a our own ,8 as wel


Conventional wisdom and comparative genomics both suggest that the human sense of smell pales in comparison to the olfactory skills of our canine and rodent relatives. Humans and mice have hundreds olfactory receptor genes, but mutations in our olfactory genes have turned the vast majority into nonfunctional “pseudogene” relics. Nonetheless, humans can detect as many as 10,000 different odors, although individual olfactory sensitivities are highly variable. About 2 million people in the United States have no sense of smell , though “specific anosmia”—insensitivity to a particular odor—is more common. One in ten people reportedly can't smell hydrogen cyanide (a poisonous gas), while 12% of participants in one study failed to detect musky odors, common perfume ingredients. An estimated one in 1,000 lucky souls can't smell butyl mercaptan, the rancid issue of skunks. At the opposite end of the spectrum are the hyperosmics, who can detect minute traces of odors such as androstenone .General anosmia is rarely genetic and more commonly results from head injury, allergy, or nasal infection. Specific anosmia is thought to arise from mutations in olfactory receptor genes. Embedded in the sensory neurons of the nasal epithelium, olfactory receptors transmit information about a chemosensory molecule (an odor) to other neurons in the brain's olfactory bulb. Researchers have long suspected that variations in the genes that encode olfactory receptors, which function on the front lines of odor recognition, may explain the vast differences seen in humans' ability to detect odors. And now, in a new study, Idan Menashe et al. have identified an olfactory receptor gene that confers a high sensitivity to the “sweaty” odorant, isovaleric acid.Menashe et al. explored the genetic basis of human olfactory diversity by focusing on a subset of olfactory receptor genes with widespread mutations  that disrupt their coding region and destroy their function. Because these genes coexist with their intact counterparts in the human population, in contrast to hundreds of other pseudogenes shared by all human beings , they are promising candidates for explaining human variance in odor detection. Individuals carry two alleles of a particular gene, one inherited from each parent. You could have two copies of the same allele (homozygous) or two different copies (heterozygous). The researchers reasoned that SPGs could generate millions of intact/disrupted combinations of alleles that a person can inherit at the dozens of SPG loci (their genotype), thus creating a huge diversity of olfactory sensitivities in the population. For example, a person with two active alleles might be hyperosmic to an odor, while someone with two nonfunctional pseudogenes might be anosmic.To test this possibility, the researchers recruited 377 volunteers for a “phenotype–genotype” association study. Focusing on the candidate olfactory receptor SPGs, they asked whether there was a relationship between participants' sensitivity to four selected odorants (their phenotypes) and the various genotypes. As participants sniffed vials filled with isoamyl acetate (which smells like pear or banana), isovaleric acid (the classic sweaty odorant), carvone (spearmint), and cineole , the researchers measured their detection thresholds .All the odors produced similar results in terms of the range of participants' sensitivities, with some individuals unable to detect the highest concentrations (anosmics), most people able to detect medium concentrations, and some able to detect the lowest (hyperosmics). Women in the study showed somewhat better sensitivity than the men, but age, smoking habits, and ethnic origin did not affect the results.OR11H7P. In keeping with established evidence that specific anosmia is a recessive trait , the researchers found that participants carrying two copies of the disrupted OR11H7P allele (the pseudogene) were mostly numb to low quantities of isovaleric acid. In contrast, those with heightened sensitivity to the compound were more likely to have at least one intact allele.In the genetic association analysis, looking for correlations between olfactory thresholds and olfactory receptor genotypes, the researchers found a strong signal between sensitivity to isovaleric acid and one gene, OR11H7P allele produced a response to isovaleric acid. Having found a variant to explain isovaleric acid sensitivity, the researchers wanted to make sure it could actually interact with this molecule. To do this, they used an assay designed to test the functional expression of olfactory receptors in frog oocytes. Using the intact allele, the pseudogenized allele, and three randomly chosen nondisrupted olfactory receptors, the researchers tested a range of isovaleric acid concentrations. Only the intact While this variant clearly plays a role in human olfactory variability, it doesn't explain all the variation observed in the study. The researchers also found a general olfactory effect, with individuals responding with similar acuity to all the odors. They conclude that hyperosmia is a complex trait that is likely modulated by a combination of genetic and environmental factors. It may be that genetic polymorphisms in individual receptors, combined with variations in fundamental olfactory pathways in the periphery and the brain, mediate the remarkable range of olfactory sensitivities in the human population. Thus, your ability to detect various odors might be enhanced if other aspects of your olfactory system are particularly efficient.Although the molecular and cellular processes that guide olfactory perception—from odor detection to the representation of different odors in the brain—have recently received considerable scrutiny, crucial elements still remained obscure. With the link between a gene variant and differences in human olfactory sensitivity now demonstrated, researchers can further explore the genetic basis of the first step in this enigmatic sensory pathway.


The distribution and the potential gene-gene and gene-environment interaction of selected metabolic genetic polymorphisms was investigated in relation to gastric cancer risk in an Italian population.CYP1A1, CYP2E1, mEH, GSTM1, GSTT1, NAT2 and SULT1A1 polymorphisms. Haplotype analysis was performed for EPHX1 exons 3 and 4, as well as CYP2E1 RsaI  and CYP2E1 DraI . The effect modification by alcohol and cigarette smoking was tested with the heterogeneity test, while the attributable proportion (AP) was used to measure the biological interaction from the gene-gene interaction analysis.One hundred and seven cases and 254 hospital controls, matched by age and gender, were genotyped for GSTT1 null genotype  and the SULT1A1 His/His genotype . No differences were observed for the haplotype distributions among cases and controls. For the first time an increased risk was detected among individuals carrying the *6 variant allele of CYP2E1 if ever-drinkers  with respect to never-drinkers  . Similarly, the effect of SULT1A1 variant genotype resulted restricted to ever-smokers, with an OR of 2.58 (95%CI: 1.27–5.25) for the carriers of His allele among smokers, and an OR of 0.86 (95%CI: 0.45–1.64) among never-smokers . The gene-gene interaction analyses demonstrated that individuals with combined GSTT1 null and NAT2 slow acetylators had an additional increased risk of gastric cancer, with an OR of 3.00 (95%CI: 1.52–5.93) and an AP of 52%.Gastric cancer risk was found to be associated with the inheritance of GSTT1, SULT1A1 and NAT2 polymorphisms appear to modulate individual's susceptibility to gastric cancer in this Italian population, particularly when more than one unfavourable genotype is present, or when combined with cigarette smoke. The increased risk for the carriers of CYP2E1*5A or *6 alleles among drinkers need to be confirmed by larger prospective studies. Helicobacter pylori infection, tobacco smoking, a high intake of salt and lack of food refrigeration all seem to play a major role .Cases and controls were interviewed by trained medical doctors using a standard questionnaire to elicit information on demographic variables, tobacco smoking  and drinking history, dietary habits and family history of cancer. Questions pertaining to lifestyle focused on the time period ending one year prior to diagnosis. Smoking status was categorized as never and ever-smokers (including both current and former smokers) and alcohol consumption as drinkers/non-drinkers . Fruit and vegetables intake was classified as high if the participant consumed at least two portions of fruit and two portions of vegetables per day. Meals salt addition was referred to the use of adding salt to the entrées during the main meals. Family history (including non-melanoma skin cancer) of cancer referred to parents, siblings and offspring. Data concerning previous GSTM1 and GSTT1 null alleles were identified using a multiplex-Polymerase Chain Reaction (PCR)-based method as described by Arand et al. pyrene-7,8-diol epoxide and smaller reactive hydrocarbons, such as ethylene oxide and diepoxybutane [SULT1A1 variant allele, who have limited detoxification capability of xeniobiotics through sulfonate conjugation, have an additional risk of gastric cancer if smokers.This is the first study conducted on an homogenous ethnic group who evaluated the effect on gastric cancer risk of several metabolic genes SNPs contemporarily, and the effect of their combination with tobacco and alcohol. One of the main source of confounding in the genetic association studies arises from population stratification, since the ethnicity itself may be related to a specific disease and to the allele frequencies as well ,31. Our y papers . IndividCYP2E1*5A or *6 alleles and gastric cancer, with an increased risk among ever-drinkers. Two previous studies [CYP2E1*5A or *6 allele and gastric cancer, however no one of them stratified data according to alcohol habits. Additionally, one study evaluating the identical association among black South-African males showed an increased risk of oesophageal cancer among drinkers carrying the CYP2E1*5A or *6 alleles [N-nitrosamines present in tobacco smoke and some dietary compounds, for which a causative role in gastric carcinogenesis has been hypothesised [5A or *6 alleles of CYP2E1 is characterized by some studies in an increased gene expression [N-nitrosamines in more reactive species, especially among drinkers since enzyme activity is induced by alcohol; ii) hyper production of reactive oxygen species and subsequent cell toxicity generated by ethanol metabolism among drinkers. We expected to gain similar results for CYP2E1 RsaI polymorphism, identically associated with increased enzyme activity, however the few subjects in the stratified analysis probably did not show it. Since these results, however, are based on very few subjects  they need to be confirmed by larger studies.To our knowledge, we reported for the first time a strong effect modification by alcohol of the association between  studies ,33 repor alleles . CYP2E1 thesised . Additiothesised . The *5Apression , so thatGSTT1 null genotype individuals contemporarily NAT2 slow acetylators have a strongly increased risk of gastric cancer, with a more than just the additive effect of the risks associated with each of the two inherited SNPs. N-acetylation is considered a major detoxification step for carcinogenic aromatic arylamines, while GSTT1 is involved in the detoxification of polycyclic aromatic hydrocarbons, so individuals with one or both depleted phase II enzyme activities might be particularly susceptible to gastric damage from carcinogens, which is supported by the finding of an additional risk for ever-smokers. We used the attributable proportion due to interaction as a measure to quantify the biological interaction between those combined SNPs and showed a strong interaction between them. Assuming that the relationships studied are causal and based on the definition of biological interaction among two component causes [GSTT1 null individuals with combined NAT2 slow acetylator phenotype are caused through a mechanism in which both risk factors are biological dependent in the same disease process. In other words, since biological interaction among two causes occurs when the effect of one is dependent from the presence of the other, in the absence of either of the two components (GSTT1 null or NAT2 slow), than a substantial number of gastric cancer cases would not occur. Given that in our population 25% of cases had a combination of those unfavourable genotypes, this means that a non negligible proportion of gastric cancer cases would have never developed if those enzymatic activities were adequate.Among the main results of our study, we found that t causes ,37, our GSTT1, SULT1A1 and NAT2 polymorphisms may modulate an individual's susceptibility to gastric cancer, particularly when more than one unfavourable genotype is present and in combination with cigarette smoke. Additionally, we showed that individuals carrying the *5A or *6 alleles of CYP2E1 are at increased risk for gastric cancer in drinkers. Clearly, since our study is based on a limited number of cases, it is critical that larger prospective studies possibly based on a single ethnic group confirms our results.This study suggests that in this Italian population, AP = attributable proportion; CI = Confidence Interval; CYP = Cytochrome P450; GST = glutathione S-transferase; HWE = Hardy-Weinberg Equilibrium; mEH = microsomal Epoxide Hydrolase; NAT = N-acetyltransferase; OR = Odds Ratio; PCR = Polymerase Chain Reaction; RR = Risk Ratio or Rate Ratio; SULT = Sulfotransferase.The author(s) declare they have no competing interests.All authors read and approved the final manuscript. SB and GR conceived the study and coordinated the research group; SB and FG performed the statistical analysis and drafted the manuscript; FAST and GLT participated in the data analysis; DA carried out the genotyping; CMVD helped to draft the manuscript; RP, SR, DDU and ALG enrolled the patients and interviewed all them.The pre-publication history for this paper can be accessed here:


Sir,We thank the correspondents for their interest in our study (Controls in the four Nordic centres in our study were recruited from population registers and from the UK centres were selected from general practitioner practices. The latter are a representative source of population-based controls as approximately 98% of the UK population is registered with a GP .With exception of Finland, where interviews were nearly all at a hospital, interviews were carried out at a location chosen by the participant, with interviewers travelling to these locations, and at a time of interview most convenient to the participant, to make the interview as stress-free as possible. Blinding interviewers to participants' case–controls status is impossible in personal interviews, but interviewers were trained to treat everyone equally and information on mobile phone use was collected with a highly structured and standardized computer-assisted interview. Exposure indices were calculated with computer programs regardless of case–control status. Excluding the small proportion of subjects who were interviewed over the telephone, mainly in Norway, did not materially affect the results. Contrary to the correspondents' assertion, the Northern part of Sweden was not part of the study region of the Swedish study. Patients or controls at distant addresses in the study were not omitted from the study.With regard to Table 4 in our paper, we refer Professors Hardell and Mild to the Methods section and the table's footnote describing the definition of the reference groups and the inclusion of bilateral phone users in both the ipsilateral and contralateral analyses.We did not include cordless phones in our analysis because they have a lower power output than mobile phones , but sevProvision of funds to the study investigators was via a firewall and was governed by legally binding agreements that guaranteed the study's complete scientific independence. The research was initiated, conducted and published without reference to any of the funding agencies. It is hard to see how a contractual clause that the funders may be informed a maximum of 7 days before publication of the results under strict terms of confidentiality infringes on this.Dr Hocking points out that misclassification in exposure assessment could have diluted a possible increased risk of acoustic neuroma among mobile phone users. We explain, in the paper, that misclassification could have diluted any real trend of risk with cumulative number of calls or hours of use, but as Dr Hocking points out himself, information on recall of year of first phone use is less likely to be seriously erroneous. Part of the reason for our conclusion of ‘no substantial risk in the first decade after starting mobile phone use’ was exactly this information.Dr Milham focuses on the decreased odds ratios in relation to recent phone use, and the possibility of control selection bias. We had discussed in our paper the potential for this bias, but also the potential for other biases, some of which might explain the decreased relative risks after recent use. As there is more than one explanation for a diminished relative risk after short-term use, and uncertainty on the extent and presence of the various biases affecting the relative risk, we do not think that one can justifiably draw any conclusions about a ‘future epidemic’, but rather, as we stated, that ‘an increase in risk after longer-term use or after a longer lag period could not be ruled out’.


A new online tool allows mapping of the different classifications of mild cognitive impairment. Despite intensive use of the term mild cognitive impairment (MCI) to describe an intermediate stage of cognitive decline between normal and pathological brain ageing, no formally agreed process of characterising this condition exists . VariousAs an opportunity for identifying individuals at risk of developing dementia, MCI is an important concept. Yet lack of consensus criteria has lead to debate about the utility of MCI, resulting in calls for abandoning its diagnosis and adopting an alternative nosology . ConsensThe first step to coding MCI is to determine the necessary criteria and thresholds for operationalisation of each definition. We compiled a comprehensive list of those classifications which represent different aspects and definitions of MCI. The necessary components for each were abstracted and formulated into a diagnostic algorithm.The main problem encountered was that while some classifications have specific criteria for implementation . If an individual fails to meet the specified outcome for a given criterion they are excluded from further mapping.In reading the flow diagram, the classification arrived at depends on the direction of decision at each criteria , you next move to the “Long-Term Memory Problem” box, and if the individual shows a long-term memory problem with intact recent memory (as indicated by the green arrow), you then arrive at a classification of BSF.  takes just three steps: from the In contrast, to arrive at a classification of A-MCI, seven steps are needed: (1) the individual is not demented (as indicated by the red arrow); (2) there is an objective memory impairment ; (3) the individual or an informant complains of memory loss (as indicated by the green arrow); (4) there is no impairment in activities of daily living (as indicated by the red arrow); (5) there is no impairment of general cognitive functioning (as indicated by the red arrow); (6) the individual has no underlying medical or neurological condition (as indicated by the red arrow); and (7) there is no impairment in another non-memory domain (as indicated by the red arrow). (See http://www.cfas.ac.uk/mciprogram/). The clinical utility of the concept of mild cognitive impairment depends on the validity of the diagnosis and the ability to predict higher rates of progression to dementia. The advantage of retrospective definition of these concepts is that they are not adjusted by current knowledge or changing criteria, an unfortunate downside of consensus criteria being that they can be influenced by changing knowledge over time. The disadvantage of mapping with retrospective information is that it removes clinical experience from the definition, and the information measured from all individuals must have enough scope to encompass the entire original definition.The flow diagram details this process for each of the 18 different classification systems, allowing for reproduction across studies. Each concept has been detailed in turn with a criteria-specific graph and the appropriate references on the CFAS Web site  exclusion criteria. However, this solution assumes that within these criteria there is one that is the best for identification of at-risk individuals. Furthermore, definitions, particularly those of objective cognitive impairment, depend on arbitrary and varying thresholds, frequently with no reference to specific values, methods, or screening measures. In retrospective studies, the mapping of these thresholds will primarily be constrained by study design, though the use of different thresholds can be used to determine the most optimal threshold value to accurately distinguish those individuals at high risk of dementia from those with low dementia risk.It is time to re-examine the concept of MCI. The diagnostic disparity and the lack of consistency in case definition calls into question what exactly is being captured in each classification. This is a fundamental weakness of research on MCI, as highlighted by the complicated nature of


SPINK1 polymorphisms in chronic pancreatitis (CP) are undefined. We hypothesize that CP occurs as a result of multiple pathological mechanisms (pathways) that are initiated by different metabolic or environmental factors (etiologies) and may be influenced differentially by downstream genetic risk factors. We tested this hypothesis by evaluating the differences in effect size of the high risk SPINK1 N34S haplotype on CP from multiple etiologies after combining clinical reports of SPINK1 N34S frequency using meta-analysis.The complex interactions between recurrent trypsin-mediated pancreatic injury, alcohol-associated pancreatic injury and SPINK1 N34S in CP  using reported etiological factors as surrogates for pathways and multiple meta-analyses to determine the differential effects of SPINK1 N34S between alcoholic and non-alcoholic etiologies. Using estimates of between-study heterogeneity, we sub-classified our 24 studies into four specific clusters. We found that SPINK1 N34S is strongly associated with CP overall , but the effect of SPINK1 N34S in alcoholic CP  was significantly smaller than in idiopathic CP  or tropical CP . Studies analyzing familial CP showed very high heterogeneity suggestive of a complex etiology with an I2 = 80.95%.The Pubmed and the Embase databases were reviewed. We studied 24 reports of The small effect of SPINK1 N34S in alcoholic subjects suggests that CP is driven through a different pathway that is largely trypsin-independent. The results also suggest that large effect sizes of SPINK1 N34S in small candidate gene studies in CP may be related to a mixture of multiple etiologic pathways leading to the same clinical endpoint. Chronic pancreatitis (CP) is a common chronic inflammatory syndrome of the pancreas that has been defined by clinical signs and symptoms linked to end-stage pathological criteriaCP is defined by the presence of chronic inflammatory cells within the pancreas, progressive fibrosis, sclerosis and parenchymal atrophy CP is sub-classified according to epidemiologic risk factors , although the relative risk of recognized environmental factors may be diminutive PRSS1) CFTR) SPINK1) SPINK1 gene mutations are thought to diminish protection against prematurely activated trypsin, and are thereby linked to trypsin-related pancreatic injury Several recent human genetic studies have provided insight into components of the pathogenic mechanisms leading to human CP, all of which are related to failure to regulate intrapancreatic trypsin activity and the associated pancreatic injury. The three primary susceptibility genes for CP include the cationic trypsinogen gene  activation through recurrent trypsinogen activation and inadequate trypsin inhibition by SPINK1. The basic model is illustrated in PRSS1 and CFTR mutations) while SPINK1-independent pathways would include factors that drive PSC to produce fibrosis through mechanisms that are generally independent of recurrent trypsinogen activation . Although a number of functionally different risk factors have been statistically associated with alcoholic CP The high-risk SPINK1 N34S in human CP, we employed meta-analysis in a manner illustrative of the growing realization that meta-analysis is most effective in examining relationships in complex diseases by evaluating between-study heterogeneity SPINK1 N34S haplotype is pathway-dependent by reviewing the world literature and gathering data from case-control studies that evaluated the association of SPINK1 N34S with CP. When possible, we reclassified patients by reported etiology and performed a series of meta-analyses on each category to determine the effect size of the high-risk SPINK1 N34S haplotype within each etiologic sub-classification. We hypothesized that etiology-based categories defining a trypsin-dependent pathway would be associated with a larger SPINK1 N34S effect than those that were trypsin-independent.In order to address questions of heterogeneity, and specifically the role of alcohol and SPINK1 polymorphisms. Only case-control studies were considered. When possible, subjects were classified according to reported etiology, with the following categories as presented in We systematically reviewed the world's literature on SPINK1 N34S high-risk haplotype in pancreatitis that were published prior to May 2007 were identified by searching the PubMed and the EMBASE databases. Search terms included polymorphism(s), SPINK1, Serine Protease Inhibitor Kazal type 1, PSTI, N34S and pancreatitis. The reference list of citations in the identified publications was reviewed to identify additional published articles not indexed by the major databases. Genetic association studies that reported the frequency of the SPINK1 N34S high-risk haplotype in patients with acute and/or chronic pancreatitis and in a control population were selected. When more than one published report used data from the same case and control population, we included only the largest study with extractable data in the meta-analysis. Corresponding authors were contacted in some cases to clarify issues of possible data duplication. Only studies that used validated genotyping methods, such as direct gene sequencing, polymerase chain reaction paired with restricted fragment length polymorphism and denaturing gradient gel electrophoresis, were included. Studies based on linkage results (pedigree studies), case reports, editorials, review articles and studies published in a language other than English were excluded.The literature search and study review was performed by two separate authors . Genetic association studies of the Data abstraction was performed by two separate authors  and differences were resolved by discussion. The data elements included the first author, journal, year of publication, country of origin, racial background of the study population (when mentioned), demographics, reported etiology of CP  and the number of cases and controls. Allelic frequency, genotypic distribution and genotyping methods were recorded. All statistical analysis was based on the number of alleles–as opposed to number of patients, in order to better quantify homozygous cases.SPINK1 N34S high-risk haplotype with CP separately. A preliminary meta-analysis combining all studies regardless of etiology was conducted. We then conducted a series of meta-analyses assessing the degree of risk of various categories of CP with SPINK1 N34S. Effect size was expressed as an OR with the corresponding 95% confidence interval (CI). Heterogeneity between studies was tested using the Cochran Q statistic and the I2 value. Because all statistical tests for heterogeneity are weak, we also included the 95% confidence interval for I22 value of 50 was considered the threshold, above which studies were considered too heterogeneous to be combined. The Haldane continuity correction (adding 0.5 to each cell) was used if the quantity of N34S-containing genotypes was equal to zero in either the cases or the controls. The presence of publication bias and small study effects were assessed using the Egger regression asymmetry test for funnel plot and the Begg-Mazumdar adjusted rank correlation test. Statistical significance was considered at a p-value of ≤0.05. In cases where the polymorphism was not detected in either cases or controls, the study was omitted from the meta-analyses as it carried a relative weight of zero. All meta-analyses were conducted using Stata version 8.2  and Comprehensive Meta-Analysis version 2.0 .For each study, we initially evaluated the association of SPINK1 N34S polymorphism with CP were identified (entified . Two stu2 = 43.97%). The random effect model showed a combined OR of 11.00 (95% C.I. = 7.59–15.93). Both the Egger and the Begg-Mazumdar tests were not statistically significant with p = 0.65 and p = 0.94 respectively.SPINK1 N34S polymorphism and alcoholic CP. The SPINK1 N34S high-risk haplotype was reported in 49 of 1,474 patient alleles and in 37 of 4,066 control alleles. There was no heterogeneity detected . Both the fixed and the random-effect model showed a pooled OR of 4.98 (95% C.I. = 3.16–7.85)–the lowest among the different categories that we analyzed. We identified patients with alcoholic CP from 9 studies . Overall, alcohol was the etiologic factor leading to chronic pancreatitis in 31% of the patients that we were able to classify. Six of nine studies failed to identify a statistically significant association between the 2 = 47.56%. The pooled OR calculated using the random-effect model was 19.15 (95% C.I. = 8.83–41.56). Four studies assessed patients with tropical pancreatitis . The high-risk haplotype was detected in 168 of 702 patient alleles and in 44 of 1,946 control alleles. The heterogeneity testing showed Q = 5.72, df = 3, p = 0.13, ISPINK1 N34S (2 of 37.98%. The pooled OR was 14.97 (95% C.I. = 9.09–24.67).Fourteen studies were included in this analysis , only two of which did not detect a statistically significant association with NK1 N34S . The CocSix studies  were analyzed. Significant heterogeneity was detected by the Cochran Q statistic . Additionally, the I-squared value was 80.94%. Due to the high degree of heterogeneity, these studies were not combined.SPINK1 N34S haplotype, and CP, and to assess the strength of any association using etiology-based classifications from previously reported studies using meta-analyses. As expected SPINK1 N34S in subpopulations, in some cases, revealed low variance. This finding suggests that these subpopulations are associated with more homogeneous pathological mechanisms. The different ORs and, occasionally, non-overlapping CIs  suggest that the CP syndrome encompasses several patient subpopulations, and may indicate that the pathologic pathway linking the proximal etiology to the PSCs in fibrosing CP are regulated to different degrees by the activity of SPINK1, as envisioned in As stated in a recent article on meta-analysis: “It is well appreciated now that besides estimating summary effects, estimation and, if possible, explanation, of the between-study heterogeneity is a very important goal for meta-analysis”When meta-analysis is applied to complex genetic syndromes, the measured effect size of a genetic variable between studies is often widely discrepant Another potential explanation is that there are multiple sub-populations with different complex, multi-step etiologic pathways that all lead to the same phenotype (e.g. end-stage organ fibrosis). Thus, a genetic factor that is critical in one etiologic pathway will only be shown to have a large effect size in populations in which that pathways dominates SPINK1 N34S with the risk of alcoholic CP was significant, but the lowest of the subgroups. This observation suggests that while a portion of the insult may be associated with premature trypsinogen activation, alcohol's primary effects seem to be via SPINK1- independent pathways  in patients with TP than controls CTSB mutations may increase the risk of inappropriate trypsin activity within the acinar cells. This may be one of several critical factors contributing to the etiology of TP.The pathway leading from an unknown factor in tropical pancreatitis to PSC activation and fibrosis is more strongly affected by SPINK1 N34S polymorphism as evidenced by a pooled OR of 14.66. Noone et al. reported a high incidence of CFTR mutations in patients with ICP and further demonstrated that the combined risk of CFTR mutations and SPINK1 polymorphisms was multiplicative rather than additive PRSS1 and CFTR mutations and the large effect of SPINK1 polymorphisms with progression to CP is consistent with our proposed model. Of note, the highest reported OR of any study (OR = ∼80) was that of Witt et al that was conducted in a relatively homogenous group of children with early onset idiopathic CP where few, if any, environmental exposures played a role, especially tobacco smoking and alcohol Patients with idiopathic CP had a significant association with the SPINK1 N34S, but these data should be interpreted with caution. Hereditary pancreatitis is an autosomal dominant disorder with very high penetrance that is caused by gain-of-function mutation in the cationic trypsinogen gene SPINK1 mutations. In this case, the etiology-based classification system is biased toward an association with SPINK1. These factors likely contribute to the high heterogeneity of effects in this classification.The risk of familial and hereditary CP is strongly associated with SPINK1 N34S effects. Furthermore, the effect of ethnicity and race was usually not reported, although the country of origin was available , thereby initiating the fibrosing process vailable  and the SPINK1 N34S polymorphism in CP confirms a significant overall pathologic association, although the reported effect size varies significantly depending on the etiology of CP. Modeling CP as a complex syndrome resulting from various pathogenic pathways with or without recurrent trypsinogen activation allows for the direct assessment of the effect of SPINK1 polymorphisms in the development of CP. While the subgroup analysis needs to be interpreted with caution, our results suggest that much of the variance in reported ORs between small studies of candidate genes in complex disorders may be attributed to a mixture of multiple etiologic pathways leading to a single clinical endpoint (e.g. organ fibrosis in CP). Alcohol appears to drive fibrosis in alcoholic CP primarily through a trypsin-independent pathway as reflected by the significantly lower association of SPINK1 N34S with this etiology. Additional studies are warranted to further assess the presence of any confounding or modifying factors and to elucidate the various pathophysiologic mechanisms involved and their implications in the etiology of CP.In conclusion, meta-analysis of association studies examining the


End-stage renal disease rates rose following widespread introduction of high fructose corn syrup in the American diet, supporting speculation that fructose harms the kidney. Sugar-sweetened soda is a primary source of fructose. We therefore hypothesized that sugary soda consumption was associated with albuminuria, a sensitive marker for kidney disease.Design was a cross-sectional analysis. Data were drawn from the National Health and Nutrition Examination Survey (NHANES), 1999–2004. The setting was a representative United States population sample. Participants included adults 20 years and older with no history of diabetes mellitus ; after exclusions for missing outcome and covariate information , the analysis dataset consisted of 9,358 subjects. Exposure was consumption of two or more sugary soft drinks, based on 24-hour dietary recall. The main outcome measure was Albuminuria, defined by albumin to creatinine ratio cutpoints of >17 mg/g  and >25 mg/g . Logistic regression adjusted for confounders . Interactions between age, race-ethnicity, gender, and overweight-obesity were explored. Further analysis adjusted for potential mediators: energy intake, basal metabolic rate, obesity, hypertension, lipids, serum uric acid, smoking, energy expenditure, and glycohemoglobin. Alternative soda intake definitions and cola consumption were employed.2, OR = 2.15 . Adjustment for potential mediators and use of alternative definitions of albuminuria and soda consumption did not appreciably change results. Diet sodas were not associated with albuminuria.Weighted albuminuria prevalence was 11%, and 17% consumed 2+ sugary soft drinks/day. The confounder-adjusted odds ratio for sugary soda was 1.40 . Associations were modified by gender (p = 0.008) and overweight-obesity (p = 0.014). Among women, the OR was 1.86 ; the OR among males was not significant. In the group with body mass under 25 kg/mFindings suggest that sugary soda consumption may be associated with kidney damage, although moderate consumption of 1 or fewer sodas does not appear to be harmful. Additional studies are needed to assess whether HFCS itself, overall excess intake of sugar, or unmeasured lifestyle and confounding factors are responsible. The rising incidence of End Stage Renal Disease (ESRD)in the United States over the past three decades is only partially attributable to increasing prevalence of recognized risk factors, most prominently diabetes mellitus Kidney disease exacts an extensive human and economic price through both ESRD and greatly elevated cardiovascular disease risk Three mechanisms may relate soda consumption to albuminuria. First, as noted, the low cost of HFCS makes it profitable for manufacturers and inexpensive for consumers, encouraging consumption of large amounts of soda. Over-consumption of sugars in any form may lead to the development of subclinical diabetes, which may eventually manifest as kidney damage years before clinical diagnosis and the onset of overt diabetes Therefore, we sought to determine whether sugary soft drink consumption was associated with albuminuria, a sensitive marker of early kidney damage, in the general population.Data were drawn from the U.S. population-based National Health and Nutrition Examination Survey (NHANES), 1999–2004. The NHANES is a complex, nationally representative sample of the entire non-institutionalized United States population, now conducted in continuous bi-annual waves by the Centers for Disease Control and Prevention 2 lower (p = 0.008).Albuminuria, the outcome variable in the primary analyses, was defined by sex-specific albumin to creatinine ratio (ACR) cutpoints of ≥17 mg/g in males and ≥25 mg/g in females; following Mattix and colleagues, lower sex-specific cutpoints for males were chosen based on their higher urinary creatinine concentrations for any given level of albumin excretion svy commands  according to NHANES analytical guidelines http://www.cdc.gov/nchs/data/nhanes/frequency/paqiaf_doc.pdf for more detail). Hypertension was a dichotomous variable defined according to Seventh Joint National Committee guidelines as systolic blood pressure ≥140, diastolic blood pressure ≥90, self-reported history of hypertension and/or use of antihypertensive medication All analyses were weighted by mobile examination clinic (MEC) weights to account for the complex survey design, and analyzed using STATA version 9.2 2, “obese”). Significant interaction was defined at the p = 0.10 level, indicating effect measure modification and warranting subgroup analyses. Because serum uric acid levels have been associated with fructose consumption In order to investigate effect measure modification of associations, interaction terms were introduced into the model between consuming 2 or more sugary sodas and indicator variables for the following: gender ; self-reported race-ethnicity by pre-defined categories ; age; and by overweight/obesity category  and column (energy intake quartile within BMI categories) differences in proportion in sugary soda intake; if there are significant biases by BMI category, then χ2 p-values for rows should be significant. We expect column χ2 p-values to be significant, since sugary soda consumption should rise with increasing total daily energy intake. nd quartile of energy intake (p = 0.035); nevertheless, the pattern is for greater sugary soda consumption to be reported among underweight and obese subjects, which is not consistent with the prediction that underreporting would be greatest in the obese subjects Several sensitivity analyses were conducted. Because 24-hour recall is an imperfect measure of caloric intake and has known biases by BMI 2), and a cutpoint with greater specificity (eGFR <45 ml/min/1.73 m2) as suggested by Go In order to determine that the results of Models 1 and 2 were not due to macroalbuminuria, we excluded those subjects with ACR≥300 mg/g . We then used linear regression with log-transformed urinary albumin as the dependent variable, and soda consumption (dichotomized as above) as the main exposure of interest, adjusting for age, race, ethnicity, gender, poverty, and the reciprocal of urinary creatinine; we modeled the log of albumin concentration, rather than ACR, as the dependent variable because the latter approach may introduce spurious correlation The study population was distributed evenly by gender and was predominantly non-Hispanic White (55.6%); due to oversampling, 25.5% of the study population was African-American, and 33% Hispanic. Summary statistics, overall and by albuminuria status, are reported in Using the “basic” Model 1, adjusting for consumption of 2 or more diet sodas per day, age, race-ethnicity, gender, and living below twice the poverty level , there w2), consumers of 2 or more sugary sodas had 2.15 times the odds of albuminuria as those who consumed 0–1 sugary drinks . Associations in other subgroups were weak and imprecise.There was no interaction between sugary soda consumption and age (p = 0.14), black or Hispanic race-ethnicity , continuous serum uric acid level (p = 0.52), or hyperuricemia (p = 0.35). There was a significant negative interaction between sugary soda consumption and being overweight (p = 0.005) or obese (p = 0.02) and with male gender (p = 0.008), indicating the need for subgroup analyses. Among women, the OR was 1.86 . In the group with low-to-optimal body weight (BMI 17.5–25 kg.m2 (the midpoint of the lowest decile of BMI), the odds ratio was 2.48 , while at a BMI of 40.2 (the midpoint of the highest decile), the OR was 0.97 . We conclude that sugary sodas are most strongly associated with albuminuria at the low end of body weight.The finding of negative interaction between obesity and sugary soda consumption was unexpected, warranting further investigated of the relationship between BMI, soda consumption, and albuminuria. We conducted a stratified analysis, fitting Model 1 within categories of underweight, optimal weight, overweight, and obesity. Because the relationship between sugary soda consumption and albuminuria appears to be modified in a quadratic manner , we addeWe then categorized sugary soda consumption as sugary cola, sugary non-cola, diet cola, and diet non-cola, with the reference being all other beverages. The strongest associations appeared for consuming 2 or more sugary non-colas per day . Sugary cola, diet cola, and non-cola consumption were not associated with albuminuria. Adjusting for basal metabolic rate (BMR) yielded comparable results to Model 1; energy intake adjustment yielded similar results to model 1 , whether or not BMR was also included in the model. Defining high-intake consumers by top quintile of sugar intake from soda yielded attenuated results yet a slight improvement in precision .2) was substituted as the dependent variable in model 1 (p = 0.347). When the CKD cutpoint was changed to <45 ml/min/1.73 m2, the odds ratio increased to 2.82 , indicating that there may be an association between sugary soda and more advanced CKD.Sensitivity analyses yielded robust results. Excluding subjects with macroalbuminuria had no appreciable effect on the estimates. When we fit a linear regression model with log-transformed urinary albumin as the outcome, we obtained results consistent with Model 1: consumers of 2 or more sugary soft drinks having on average 1.13 mg/L higher albumin concentration than consumers of 0–1 drinks . Interaction terms with 2+ sugary drinks were significant for gender (p = 0.03), and for overweight (p = 0.01) but not for obesity (p = 0.42). Furthermore, log-transformed urinary albumin concentration was 1.09 mg/L higher among drinkers of 2 or more sugary colas , and 1.23 mg/L higher among sugary non-cola drinkers , than among non-soda drinkers; neither diet colas nor non-colas were related to albuminuria. Modeling continuous eGFR as a function of the covariates used in Model 1 did not yield statistically significant association with sugary soda consumption (p = 0.586); there was also no association when Stage 3 CKD  their energy intake according to perceived social norms Optimal body weight may increase susceptibility because obesity is a competing mechanism by which sugar consumption could cause kidney damage Our results suggest that fructose may be nephrotoxic via pathways other than diabetes, long-term blood glucose level, hypertension, or obesity, because adjusting for these potential mediators did not eliminate the association. We caution that mediator-adjusted models are not causal models, and they rarely meet the conditions necessary to accurately identify independent effects of the main exposure Recent reports have shown that fructose consumption in the form of soft drinks increases the risk of kidney stones and gout Several limitations must be noted. First, this study was cross-sectional, precluding us from observing longitudinal associations. In particular, we could not determine if the complex interaction between BMI, soda consumption, and albuminuria was confounded by processes such as wasting associated with chronic disease 2 was stronger than the association with albuminuria, which may reflect an association with more advanced chronic kidney disease In spite of these limitations, several strengths deserve mention. First, we used nationally representative data drawn from the NHANES surveys, yielding population-based measures of association that are generalizable to the general population, including racial and ethnic minorities. Second, because we had over 9,000 subjects and over 1,000 cases, we could explore effect measure modification (interactions) and estimate associations within subgroups of participants. Third, the richness of the NHANES dataset allowed us to adjust for a host of potential confounders and mediators, and associations with sugary soda consumption remained elevated even after adjusting for these factors. We conducted several sensitivity analyses, including modeling the log of albumin excretion, use of energy intake to define soda consumption, and adjustment for overall energy intake and basal metabolic rate. The associations were robust to different definitions of exposure, outcome, and adjustment factors. Finally, we found that the association of sugary soda consumption with eGFR below <45 ml/min/1.73 mIn conclusion, we have found that sugary soft drink consumption is associated with albuminuria. While these results are consistent with prior knowledge, they can only suggest that HFCS plays a causal role in kidney disease. Longitudinal studies, with measures of HFCS and other sugar consumption, are needed to formally test this hypothesis. At this point in time, policy recommendations regarding soda consumption or HFCS would be premature. Our findings should be seen in the context of disparate associations with CKD and metabolic syndrome that have recently been found for colas (but not sugary non-colas) and diet sodas