Selective breeding programmes, based on prion protein (PrP) genotype, have been introduced throughout the European Union to reduce the risk of sheep transmissible spongiform encephalopathies (TSEs). These programmes could have negative consequences on other important traits, such as fitness and production traits, if the PrP gene has pleiotropic effects or is in linkage disequilibrium with genes affecting these traits. This paper presents the results of an investigation into associations between lamb survival and PrP genotype in ten mainstream sheep breeds in Great Britain (GB). In addition, the reasons for lamb deaths were examined in order to identify any associations between these and PrP genotype.Survival times from birth to weaning were analysed for over 38000 lambs (2427 dead and 36096 live lambs) from 128 flocks using Cox proportional hazard models for each breed, including additive animal genetic effects. No significant associations between PrP genotype and lamb survival were identified, except in the Charollais breed for which there was a higher risk of mortality in lambs of the ARR/VRQ genotype compared with those of the ARR/ARR genotype. Significant effects of birth weight, litter size, sex, age of dam and year of birth on survival were also identified. For all breeds the reasons for death changed significantly with age; however, no significant associations between reason for death and PrP genotype were found for any of the breeds.This study found no evidence to suggest that a selective breeding programme based on PrP genotype will have a detrimental effect on lamb survival. The only significant effect of PrP genotype identified was likely to be of little consequence because an increased risk of mortality was associated with a genotype that is selected against in current breeding strategies. Large-scale selective breeding programmes have been introduced throughout the European Union to reduce the risk of sheep transmissible spongiform encephalopathies (TSEs) and, in particular, the risk to human health posed by the possible presence of bovine spongiform encephalopathy (BSE) in sheep . These pConcerns have been expressed, however, that such large-scale breeding programmes based on one specific factor  could have detrimental side-effects on sheep performance. These concerns stem from farmers' assertions that sheep carrying high-risk genotypes are more hardy or perform better than their flock-mates which carry low-risk genotypes ,6, and fTo date there have been numerous studies examining associations between production traits and PrP genotype -14, noneIn this paper, we report the results of an investigation into associations between lamb survival and PrP genotype in ten mainstream sheep breeds in Great Britain (GB). These breeds were drawn from all sectors of the British sheep industry, from hardy hill breeds to those raised in less hostile environments, and include some of the most important commercial breeds. The aim of the study was not to estimate or compare absolute lamb mortality in the different breeds, rather it was to identify factors influencing relative mortality within individual breeds, in particular, PrP genotype. In addition to lamb survival, we also examined the reason for lamb deaths in order to identify any associations between these and PrP genotype.The ten sheep breeds included in the study Table  were selFarmers were eligible to join the study if they recorded their flock performance traits using the Meat and Livestock Commission's Signet Sheepbreeder, a genetic evaluation programme, and were members of the Ram Genotyping Scheme (RGS) of the National Scrapie Plan for GB (NSP). All eligible breeders were invited to join the study by letter from Signet. In addition, the study was promoted amongst farmers by telephone and direct contact at breeder meetings. Farmers provided flock details and signed consent forms to release their Signet and NSP data to research partners in the study.® ear tags (Agribiogen Biotechnologie GmbH). Samples were sent for PrP genotyping using proprietary commercial technology to Cellmark, part of Orchid Biosciences Europe Ltd, under contract to Innovis Ltd (formerly CBS Technologies). Results were collated by Innovis Ltd and provided to the Veterinary Laboratories Agency (VLA). Live lambs were blood sampled by Animal Health (formerly State Veterinary Service) as part of the NSP on special visits to participating flocks each year. Blood samples were processed and PrP genotyped using proprietary commercial technology by Cellmark (70%) or LGC (30%). All live lambs on farm were sampled, with visits encouraged to take place before replacement lambs had been selected for use in the flock and before lambs were sent for sale or slaughter.Tissue samples were taken from dead lambs found by the farmers using TipyfixFull details of the PrP genotypes included in the study for each breed are presented in the additional material , litter size born, litter size reared and fostering information for all lambs born in participating flocks. Genotype data for live animals were provided to Signet by the NSP Administration Centre following blood sampling visits to each farm. The genotypes were matched to Signet data using the ear tag provided on the day of sampling. These data were subsequently provided to the VLA via a regular electronic transfer of animal data each year.Data on dead lambs were collected using a submission form, which was linked by barcode to the tissue sample sent for PrP genotyping. Data captured on the form included ear tag number, date of birth, date of death, dam identity, sex and the breeder's opinion of the reason for death. Submission forms were sent to VLA, where the data were double-entered onto a database. The results of the tissue genotyping were returned to Innovis by Cellmark and provided electronically to the VLA, where the PrP genotype was matched by barcode to individual animals.The number of records in each data-set ranged from around 800 lambs for the Bluefaced Leicester to around 10000 lambs for the Texel, and the number of dead lambs varied from 71 for the Beulah Speckled Face to 520 for the Charollais (Table 2) in the range 0.2 to 0.4 for three breeds ; however, the variance estimate was close to zero (<0.01) for the remaining seven breeds. This is a consequence of the relatively small number of mortalities available in several of the breeds and the fact that lamb survival is a lowly heritable trait, particularly if only a subsample of all mortalities are recorded. Consequently, the animal-level variance was fixed at σ2 = 0.2 for all breeds rather than being estimated; this is similar to estimates obtained in previous studies of lamb survival [Lamb survival times were analysed from birth (0 days) to weaning (120 days). Weaning was chosen as the end point, because this is an age before which lambs are dispersed, for example, selected as replacements or sent for slaughter or sale. A total of 204 lambs in the data-set died after 120 days, and these were included in the analyses as censored at 120 days. Survival times were modelled using Cox proportional hazard models , with thsurvival -21.P > 0.05) terms, starting from a model incorporating PrP genotype . The models were implemented in R [The reason for death supplied by the breeder was coded into one of five classes: lambing-associated ; misfortune ; physical ; disease; or other, including unknown. Potential associations were explored between reason for death and PrP genotype, breed, sex, age at death (coded as: birth (0 days); perinatal period (1–14 days); perinatal period to weaning (15–120 days); post-weaning (≥ 120 days)) and year of birth. Initially, a multinomial log-linear model was constructed including all the above five factors, together with pairwise interactions between breed and the four remaining factors. Model simplification proceeded by stepwise deletion of non-significant terms , and it was not replicated in the model including birth weight. Indeed no significant associations between PrP genotype and lamb survival were identified for any of the remaining breeds considered in the study, nor were there any consistent patterns in the effect of PrP genotype on lamb survival across breeds. This lack of consistency between studies suggests that if there is an association between PrP genotype and lamb survival it may be due to linkage disequilibrium rather that pleiotropy, or may not be constant across flocks.A recent study of lamb survival in two Scottish Blackface flocks found that lambs carrying the ARQ allele had higher postnatal survival than those carrying the ARR or AHQ allele . A similAlthough only one significant association between PrP genotype and survival was identified, not all PrP genotypes commonly found in each breed were represented in this study . This could reduce the power of the data-set or the precision of the results, but reflects the level of mortality in the study flocks.Birth weight has often been identified as an important factor for lamb survival -29, inclNo associations between PrP genotype and reason for death were identified in this study Table . The priThis study found no evidence to suggest that a selective breeding programme based on PrP genotype will have a detrimental effect on lamb survival, at least for the ten breeds included. Indeed, the only significant effect of PrP genotype identified here is likely to be of little consequence, because it found an increased risk of mortality in a genotype (ARR/VRQ) of low frequency that is actively selected against as part of current breeding strategies.SG performed the statistical analysis and drafted the manuscript. CC managed and collated the final data-sets and performed the statistical analysis. KH provided input to the statistical analysis. KB matched NSP genotypes to Signet records and CD managed the Signet data. ET and WH managed the collection of data on dead lambs. WH, SCB, BV and RE conceived the study and led the project. All authors critically revised the manuscript and read and approved the final version.Additional material.Click here for file


Studies in mothers of Down syndrome individuals (MDS) point to a role for polymorphisms in folate metabolic genes in increasing chromosome damage and maternal risk for a Down syndrome (DS) pregnancy, suggesting complex gene-gene interactions. This study aimed to analyze a dataset of genetic and cytogenetic data in an Italian group of MDS and mothers of healthy children (control mothers) to assess the predictive capacity of artificial neural networks assembled in TWIST system in distinguish consistently these two different conditions and to identify the variables expressing the maximal amount of relevant information to the condition of being mother of a DS child.MTHFR 677C>T and 1298A>C, MTRR 66A>G, MTR 2756A>G, RFC1 80G>A and TYMS 28bp repeats and 1494 6bp deletion). Data were analysed using TWIST system in combination with supervised artificial neural networks, and a semantic connectivity map.The dataset consisted of the following variables: the frequency of chromosome damage in peripheral lymphocytes (BNMN frequency) and the genotype for 7 common polymorphisms in folate metabolic genes  that were subsequently used to discriminate between MDS and control mothers with 90% accuracy. The semantic connectivity map provided important information on the complex biological connections between the studied variables and the two conditions (being MDS or control mother).TWIST system selected 6 variables (BNMN frequency, Overall, the study suggests a link between polymorphisms in folate metabolic genes and DS risk in Italian women. Folates are essential nutrients required for one-carbon biosynthetic and epigenetic processes. After intestinal absorption, folate metabolism requires reduction and methylation into the liver to form 5-methyltetrahydrofolate (5-methylTHF), release into the blood and cellular uptake; then it can be used for the synthesis of DNA and RNA precursors or for the conversion of homocysteine (Hcy) to methionine, which is then used to form the main DNA methylating agent S-adenosylmethionine (SAM) ,2. A diaWe observed that MDS who had a DS child in young age (35 years or before) have an increased frequency of chromosome damage and malsegregation events in peripheral blood lymphocytes, detectable by means of the micronucleus assay coupled by fluorescence in situ hybridization technique compared to women who gave birth to healthy children . Others Overall, genetic association studies as well as cytogenetic studies performed in MDS point to a possible role of folate gene polymorphisms in affecting chromosome malsegregation events, thus increasing the risk for a DS pregnancy. Moreover, they all suggest complex interactions between several factors within the folate metabolic pathway .We performed the present study using Artificial Neural Networks (ANNs) to identify key factors linking folate metabolism to chromosome malsegregation and the risk of having a DS child. The method used by ANNs aims to understand natural processes and recreate those processes using automated models. These networks allow a method of forecasting with understanding of the relationship between variables, in particular non-linear relationships -17. ANNsThis study aimed to analyze a dataset of genetic and cytogenetic data obtained from MDS and mothers of healthy children (control mothers) ,14 to asMTHFR 677C>T polymorphism , 3) genotype for the MTHFR 1298A>C polymorphism , 4) genotype for the MTRR 66A>G polymorphism , 5) genotype for the MTR 2756A>G polymorphism , 6) genotype for the RFC1 80G>A polymorphism , 7) genotype for TYMS 28bp repeats  and 1494 6bp deletion  polymorphisms. Table We aimed to re-analyze from a completely new perspective most of the data obtained from our previous studies ,14. FromThe database analyzed in the present study is provided as additional material  and validated PCR/RFLP techniques as described elsewhere ,14. All Advanced intelligent systems based on novel coupling of artificial neural networks and evolutionary algorithms have been applied. In this study we applied TWIST system  and supeData analysis was performed using a re-sampling system named TWIST developed by Semeion Research Centre. The TWIST system consists in an ensemble of two previously described systems: T&T and IS . The T&TAfter this processing, the features that were most significant for the classification were selected and at the same time the training set and the testing set were created with a function of probability distribution similar to the one that provided the best results in the classification. A series of supervised Multi Layer Perceptrons, with four hidden units, were then used for the classification task. The final ANNs which were trained and tested on the new data set generated by TWIST system are "virgin" and operate independently and blindly from each other and from TWIST system.An existing mapping method ,27 was uAfter the training phase, the weights matrix of the Auto-CM represents the warped landscape of the dataset. Subsequently, a simple filter to the weights matrix of the Auto-CM system was applied to obtain a map of the main connections between the variables of the dataset and the basic semantic of their similarities, defined connectivity map as detailed by Buscema and Grossi  and BuscThe linear correlation index between the input variables and the target variable was generally very low, with exception of BNMN‰ (r = 0.54). This gave the rationale to employ artificial neural networks.The application of TWIST system allowed the selection of a subgroup of 6 variables described in table Figure TYMS 1494 6bp +/- genotype resulted to be the principal hub of the system, i.e. the dominant variable. This variable was connected with TYMS 28bp 2R/3R, with MTR 2756AA, and with both MTHFR 677CT and MTHFR 1298AA variables. Connections were detected among MTHFR 677 and 1298 variables; particularly, MTHFR 677TT was connected with MTHFR 1298AA, whereas MTHFR 1298AC was connected with both MTHFR 677CC and MTHFR 677CT variables. Moreover, TYMS 28bp repeats and 1494 6bp deletion variables were always connected each other; particularly, TYMS 1494 6bp +/- resulted connected with TYMS 28bp 2R/3R, TYMS 1494 6bp +/+ with TYMS 28bp 2R/2R, and TYMS 1494 6bp -/- with TYMS 28bp 3R/3R. BNMN‰, RFC1 80GG and TYMS 28bp 2R/3R resulted to be the three variables connected with the condition of being MDS. On the contrary, RFC1 80AA, MTR 2756AA and TYMS 28bp 3R/3R resulted to be the three variables connected with the condition of being a control mother.The This study explored the association between 7 polymorphisms in the folate metabolic pathway, chromosome damage in peripheral blood lymphocytes (as BNMN frequency), and the condition of being mother of a DS child with complementary non-linear approaches: supervised neural networks (ANNs), and the semantic connectivity map.RFC1 80AA, MTHFR 677TT, MTR 2756AA, TYMS 1494 6bp del +/+ and TYMS 28bp 3R/3R polymorphisms resulted the most important variables for discriminating between MDS and control mothers  allele and the 2R allele are rare [TYMS mRNA, while the 2R allele is associated with reduced transcription of the TYMS gene. Therefore, their combined presence on the same haplotype could seriously impair TYMS production [Similarly, the connectivity map Figure  showed aare rare . A possioduction .RFC1 80GG genotype, the BNMN frequency, and the TYMS 2R/3R genotype. On the contrary, the RFC1 80AA genotype, the TYMS 3R/3R genotype and the MTR 2756AA genotype, are closely connected to the condition of being a control mother. The association between the RFC1 80G allele and increased risk for having a DS child has been often observed in Italian populations. We first reported a possible role for the RFC1 80G>A polymorphism, in interaction with MTHFR 677C>T and 1298A>C variants, in affecting DS risk in Italy, suggesting a causative role for the RFC1 80G allele and a protective role for the RFC1 80A one [RFC1 80GG genotype and increased risk for having a DS child in Italy [RFC1 80A allele previously observed by us [RFC1 80G allele could increase the risk for having a DS child, while the RFC1 80A allele could be protective [RFC1 80GG genotype and the condition of being MDS, as well as a connection between the RFC1 80AA genotype and the condition of being control mother.The connectivity map Figure  revealed 80A one . Subsequin Italy . They aled by us . Overallotective . InteresThe BNMN frequency is another variable connected with the condition of being MDS. Several previous studies by us ,13,14 reMTR 2756AA genotype resulted to be connected to the condition of being a control mother as well as to the MTRR 66AA genotype (Figure MTR 2756G allele and increased DS risk in Italy, arguing for a protective role for the 2756A allele. They also reported an interaction between MTR 2756A>G and MTRR 66A>G polymorphisms in increasing DS risk [MTR 2756AA and MTHFR 677TT genotypes in increasing DS risk in Italy [The e Figure . As show DS risk . We recein Italy . HoweverTYMS polymorphisms in the connectivity map. Indeed, the TYMS 6bp +/- genotype resulted to be the principal hub of the system, the TYMS 28bp 2R/3R genotype was connected to the condition of being MDS, and the TYMS 28bp 3R/3R genotype to that of being a control mother (Figure TYMS and MTHFR polymorphisms that can be explained by the following observation: given that both enzymes utilize the same substrate, polymorphisms reducing MTHFR enzyme activity might shift pools of 5,10-methyleneTHF from DNA methylation toward DNA synthesis, whereas polymorphisms affecting TYMS activity might shift the pathway from DNA synthesis toward DNA methylation [MTHFR and TYMS polymorphisms in increasing DS risk, suggesting that an impaired balance between DNA synthesis and methylation processes could favour chromosome malsegregation events [TYMS polymorphisms in DS risk.A very interesting finding of the present study is in the central role played by r Figure . TYMS shr Figure . Both TYr Figure  shows sehylation . We recen events . The resIn conclusion, the currently available literature suggests that complex interactions between polymorphisms in folate metabolizing genes might account for an increased maternal risk to have a DS child; however, given the complexity of the pathway, those complex interactions cannot be easily understood and none of the so far studied polymorphisms can be used in genetic counselling to predict the maternal risk for having a DS child . The preThe authors declare that they have no competing interests.FC: conceived and designed the study, supervised all the work related with the collection of genetic and cytogenetic data and all the work in the laboratory, prepared the database for the specific analysis, interpreted the data, wrote the manuscript. EG: carried out the ANNs and PC analysis, was involved with the drafting of the related technical parts in the manuscript including tables and figures, and revised critically the entire manuscript. FM: performed genotyping and helped preparing the database for the specific analysis. LM: supervised the research group, helped drafting and revising critically the entire manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1755-8794/3/42/prepubDatabase. The database used for analysis in an Excel .csv format.Click here for fileTwist System. The pdf file contains a detailed description of the Twist system. This document already existed as documentation [entation ,24,25 anClick here for fileAuto-CM System. The pfd file contains a detailed description of the theory behind Auto-CM system. This document already existed as documentation [entation ,27 and iClick here for file


Mefloquine has historically been considered safe and well-tolerated for long-term malaria chemoprophylaxis, but its prescribing requires careful attention to rule out contraindications to its use, including a history of certain psychiatric and neurological disorders. The prevalence of these disorders has not been defined in cohorts of U.S. military personnel deployed to areas where long-term malaria chemoprophylaxis is indicated.Military medical surveillance and pharmacosurveillance databases were utilized to identify contraindications to mefloquine use among a cohort of 11,725 active duty U.S. military personnel recently deployed to Afghanistan.A total of 9.6% of the cohort had evidence of a contraindication. Females were more than twice as likely as males to have a contraindication .These findings underscore the importance of proper systematic screening prior to prescribing and dispensing mefloquine, and the need to provide alternatives to mefloquine suitable for long-term administration among deployed U.S. military personnel. Plasmodium vivax is the principal cause. To protect against the threat of malaria, U.S. military personnel deploying to Afghanistan may be prescribed mefloquine, which must be taken continuously throughout deployments lasting as long as 15 months. Although the long-term use of mefloquine for malaria chemoprophylaxis has historically been considered safe and well-tolerated among civilian travelers [Malaria poses a continued threat to U.S. military personnel. At least 423 blood-smear confirmed cases of malaria were diagnosed among members of the U.S. military between January 1st, 2000 and December 31st, 2005; of which at least 64 represent cases attributable to service in Afghanistan [To quantify the prevalence of, and identify demographic characteristics associated with contraindications to mefloquine use among deployed U.S. military personnel, a retrospective cohort study was performed using information available in military personnel, medical surveillance ,4,7, andTo identify the study cohort, a roster was obtained comprising all active duty U.S. military personnel assigned in support of combat and reconstruction operations in Afghanistan and deployed within six months of a reference date in early 2007. Demographic covariate data on each was obtained from the Defense Medical Surveillance System (DMSS) .For each member of the study cohort, DMSS was queried for International Classification of Diseases, Ninth Revision, Clinical Modification ICD-9CM) -coded CM -coded. These iFor each member of the study cohort, Pharmacy Data Transaction System (PDTS) records  were exa2 statistics, P-values, calculation of crude odds ratios (ORs) and associated 95% confidence intervals (CIs) were performed using SAS software  [Analyses included producing summary and descriptive statistics. The prevalence and the statistical significance of associations between demographic characteristics and a contraindication to mefloquine use were determined. All statistical analyses, including producing Pearson's χary, NC) .The study cohort consisted of 11,725 subjects. A total of 558 subjects (4.8%) had a medical contraindication . A total of 837 (7.1%) had a pharmacologic contraindication (i.e. one or more prescriptions filled prior to deployment for medications consistent with a contraindication to mefloquine use). A total of 1,127 (9.6%) had either or both (i.e. at least one or more such diagnosis or at least one or more prescription)  had neurological diagnoses and 531 (95.2%) had psychiatric diagnoses. The groups were mutually exclusive. Among those with neurological diagnoses (0.2% of the study cohort), all 27 had outpatient diagnoses, and none had inpatient diagnoses. Of the 27, 13 (48%) had primary diagnoses. Among those with psychiatric diagnoses (4.5% of the study cohort), three had inpatient diagnoses, and the remainder had one or more outpatient diagnoses, of which 411 (78%) had primary diagnoses.Of the 837 with one or more prescriptions, 78 (9.4%) had a prescription for an ADHD treatment; 93 (11.1%) for an anticonvulsant; 315 (37.6%) for an antidepressant; two (0.2%) for an antiparkinsonian; 24 (2.9%) for an antipsychotic; and 446 (53.3%) for a sedative-anxiolytic. A total of 100 (11.9%) had prescriptions for two or more classes of drug.2 = 38.9, P < 0.001) and military specialty , and between the study cohort and those with prescriptions by gender , military specialty , and age category . Between the study cohort and those with either diagnosis or prescription, there were significant differences by gender , military specialty , and age category . Women had over twice the odds of having either diagnosis or prescription . Among those with any deployment history, the odds of either diagnosis or prescription was higher, but this difference was not statistically significant .There were significant differences between the study cohort and those with diagnoses by gender , self-selected for military service from the U.S. general population and subject to considerable selection pressures from commissioning, enlistment, or periodic health screenings. These pressures would ordinarily result in this population being generally more healthy than members of the U.S. civilian population, and would ordinarily be expected to reduce the prevalence of health conditions incompatible with continued military service and deployment.Interestingly, this bias, which is commonly referred to as the "healthy-warrior effect"  may not Despite the substantial differences in health histories and demographics between studies, this study finds a similar overall rate of contraindication to mefloquine use as a prior prospective study in the general population, in which 9% of 2,389 civilian travelers with available medical histories presenting to a travel medicine clinic were evaluated . SimilarThis study employed medical surveillance databases to identify an arbitrary set of ICD-9CM diagnosis codes defining a medical contraindication to mefloquine use. The diagnosis codes were selected based on the clinical judgment of the study authors. This methodology reflects the clinical uncertainty faced by individual healthcare providers in interpreting the mefloquine package insert. Of the listed contraindications to mefloquine use, the terms "major psychiatric disorders" and "history of convulsions" leave significant room for interpretation. In this study, a liberal interpretation of neurological disorder, and a relatively conservative interpretation of psychiatric disorder were employed. This had the effect of including and excluding, respectively, many diagnoses which individual providers might or might not consider a contraindication.The use of pharmacosurveillance databases to infer the presence of a contraindicating medical condition is reasonable. Previous studies  have shoThese findings underscore that caution must be exercized by healthcare providers in prescribing mefloquine to deploying U.S. military personnel, and lend strong support to policies prohibiting the practice of mass-prescribing mefloquine without an individualized review of medical records. Given the significant prevalence of its contraindications, a careful review and screening of available medical records (including available pharmacy records) and thorough counseling prior to prescribing and dispensing mefloquine to U.S. military personnel is strongly indicated. The use of existing medical and pharmacosurveillance databases to populate automated decision-support systems to assist clinicians in identifying personnel with possible contraindications may improve the sensitivity of such review and screening. The development of such systems would require that the ICD-9CM codes and medications deemed consistent with a contraindication to mefloquine use be formally defined.Plasmodium vivax infection [This study also highlights the importance of investing in the further development and testing of alternative anti-malaria drugs which retain the advantages in compliance and convenience of the weekly dosing schedule of mefloquine. For U.S. service members in whom mefloquine in contraindicated, tafenoquine may be a promising alternative , both fonfection .Although not sharing the particular contraindications of mefloquine, tafenoquine carries a significant risk of haemolysis among those with glucose-6-phosphate dehydrogenase (G6PD) deficiency syndrome. A recent study suggests that the prevalence of this contraindication among deploying U.S. military populations is lower than that of contraindications to mefloquine found in the present study, with 2.5% of males and 1.6% of females deficient for G6PD .In light of this study's findings, upon U.S. Food and Drug Administration licensure and upon the completion of studies confirming its safety and suitability for long-term administration, tafenoquine may prove to be a viable option to replace mefloquine for routine use in the U.S. military. Universal screening for G6PD deficiency upon entry to U.S. military service may be a prudent means of ensuring the safe administration of this drug and aiding in the pre-selection of individuals most suited for long-term deployment to malarious areas.ADHD : Attention-Deficit Hyperactivity Disorder; CI: Confidence Interval; DMSS: Defense Medical Surveillance System; G6PD: Glucose-6-Phosphate Dehydrogenase; ICD9-CM: International Classification of Diseases, Ninth Revision, Clinical Modification; OR: Odds Ratio; PDTS: Pharmacy Data Transaction SystemThe author(s) declare that they have no competing interests.JBC conceived of the study. PPP coordinated acquisition of DMSS data and assisted in analysis. RLN integrated PDTS, and DMSS data, performed the statistical analyses, and drafted the manuscript. All authors assisted in interpreting the findings and reviewing and editing the manuscript.The opinions expressed in this paper are those of the authors and do not reflect the official policies or positions of the Department of the Army or the Department of Defense.


Progression-free survival , TTP  and overall response rate  were significantly better for gemcitabine combination chemotherapy, but offset by the greater grade 3/4 toxicity thrombocytopenia , leucopenia , neutropenia , nausea , vomiting  and diarrhoea . There is no significant advantage on secondary end point analyses for administering 5FU in combination over 5FU alone. There is improved PFS/TTP and response rate, with gemcitabine-based combinations, although this comes with greater toxicity.In advanced pancreatic cancer, level one evidence has established a significant survival advantage with chemotherapy, compared to best supportive care. The treatment-associated toxicity needs to be evaluated. This study examines the secondary outcome measures for chemotherapy in advanced pancreatic cancer using meta-analyses. A systematic review was undertaken employing Cochrane methodology, with search of databases, conference proceedings and trial registers. The secondary end points were progression-free survival (PFS)/time to progression (TTP) (summarised using the hazard ratio (HR)), response rate and toxicity (summarised using relative risk). There was no significant advantage of 5FU combinations  Advanced pancreatic cancer has a poor prognosis, with a median survival of 2–6 months for metastatic disease and 6–11 months for locally advanced disease . Chemothvs gemcitabine.  or time to progression (TTP – time from randomisation to disease progression), overall response rate  and toxicity .Individual trial level time to event data (PFS/TTP) were summarised by the log HR and its variance was approximated using previously reported methods  (Dichotomous data (ORR and toxicity) were summarised using relative risks and 95% confidence intervals and pooled using the Mantel–Haenszel method for combining trials (ogeneity ) and intResults are presented for the comparisons with adequate data to assess the secondary outcome measures.n=700) included in this comparison. A HR of <1 indicates a survival advantage for 5FU combination chemotherapy.There were five studies  .Overall response rate appeared better in the gemcitabine arm; however, the wide confidence interval suggests a benefit for either gemcitabine or 5FU . In the Burris trial , TTP   and ORR Examination of the funnel plots revealed evidence of bias, possibly publication bias, but this is difficult to interpret in view of the small number of studies within each comparison.−2 day−1 in both arms) compared to the −2 day−1 used in the single-agent arm and 1000 mg m−2 used in the combination arm).5FU combinations did not prolong TTP over 5FU alone, despite significantly better response rate with the former. The study of As overall survival is a better indicator of efficacy than response rate (vs 5FU comparison, as these results were only available for one randomised trial.Meta-analyses of the secondary end points were not possible in the gemcitabine vs gemcitabine employed differing survival analyses methodology (Previous meta-analyses of secondary end points evaluating gemcitabine-based combinations vs gemcitabine alone, our findings of improved PFS/TTP are in agreement with the meta-analyses of et al and Milella et al (vs gemcitabine alone found better PFS/TTP in the combination arm (For gemcitabine-based chemotherapy We have done our utmost to cover most reported end points in the randomised controlled trials. We could not address quality of life due to the different methods used for reporting quality of life. Although we have pooled the response rate and adverse events data across studies to permit a clinically relevant analysis, reporting of these parameters varied. Response rates were reported using clinical parameters, the WHO and RECIST criteria, whereas the CTC, WHO and ECOG scales were used for toxicity data.To conclude, there is insufficient evidence to suggest a TTP, response rate and toxicity advantage in administering 5FU in combination with other chemotherapy agents over 5FU alone. There is a small but significant TTP/PFS advantage, as well as improved response rate, with gemcitabine-based combinations, and this provides a justification for the use of these agents, despite their greater toxicity. An area for further randomised controlled trials to assess is which gemcitabine-based combination chemotherapy regimens are least toxic, while retaining all the other advantages of the combination approach.


Coronary artery disease (CAD) is a leading cause of death worldwide and in the United States it is responsible for more than 500,000 deaths each year. Genome-wide association studies (GWAS) have revealed connections between a number of common single nucleotide polymorphisms (SNPs) and CAD and other cardiovascular diseases. The p.Trp719Arg SNP in the kinesin-like family 6 (KIF6) gene has recently been reported as a potential risk factor for CAD as well as a predictor of response to statin therapy. KIF6) gene was first identified as a potential risk factor for CAD in 2007KIF6 p.Trp719Arg testing include CAD patients with or without a family history of CAD or myocardial infarction (MI), and patients considering statin therapy.Coronary artery disease (CAD), also called coronary heart disease (CHD), is a leading cause of death worldwide, and the most common cause of death in the United States. CAD results from the accumulation of plaque within the walls of coronary arteries, leading to the limitation of blood flow to the heart. Manifestations of CAD include angina, myocardial infarction (MI), heart failure, and arrhythmia. Risk factors associated with cardiovascular disease include age, smoking status, obesity and metabolic syndrome, high low-density lipoprotein cholesterol (LDL-C) or low high-density lipoprotein cholesterol (HDL-C) levels, elevated blood pressure, physical inactivity, insulin resistance, and diabetes mellitusKIF6 p.Trp719Arg test genotypes the p.Trp719Arg SNP located within the KIF6 gene. The test is considered positive if the patient is either heterozygous or homozygous for the KIF6 p.Trp719Arg SNP. The KIF6 p.Trp719Arg test is provided by a single laboratory, Berkeley HeartLab Inc.. No specific technical description of the assay method used for KIF6 genotyping was found on the Berkeley HeartLab website,The CAD is the most common cause of death in the United StatesSystematic evidence reviewsNone identified.Recommendations by independent groupNone identified.Guidelines by professional groupsNone identified.KIF6 OR kinesin-like family 6 OR kinesin family member 6) AND (Trp719Arg OR 719Arg) AND (cardiac OR coronary OR cardiovascular). After limiting to English language, human, and published since January 1, 1999; this search yielded 7 citations. Citations from relevant references were also reviewed and included as appropriate.     A literature search of MEDLINE and EMBASE was completed on September 18, 2010, using the search terms .KIF6 p.Trp719Arg testing were identified.No studies that specifically addressed the analytical validity of One study claimed an analytical sensitivity of 97.9% using PCR and allele-specific oligonucleotide ligation Clinical Validity:Test accuracy and reliability in [supporting clinical or public health assessment] . KIF6 p.Trp719Arg testing and CAD and associated benefit from statin therapy consists of 5 retrospective genetic relationship studies published by authors associated with Celera Corp., the developer of the KIF6 p.Trp719Arg testThe current body of peer-reviewed research supporting a link betweenKIF6 p.Trp719Arg carriers (heterozygous and homozygous) across these studies ranged from 50.7% to 62.3%. All of the studies except the Women's Health Study (WHS) investigated the use of statin therapy in at-risk populations.The patient populations investigated in these studies consisted primarily of white Americans or Europeans, except for one study that included a relatively small number of nonwhite patientsKIF6 p.Trp719Arg carriers were 1.18  for CHD in the WHSKIF6 p.Trp719Arg carriersDue in part to the variation in endpoints utilized in these studies, it is difficult to draw broad conclusions from the data provided. Reported hazard ratios (HRs) for CAD for  KIF6 p.Trp719Arg carriers derive significantly more benefit from statin therapy while no significant improvement was reported in noncarriers in any study. In the first analysis of CARE, an absolute risk reduction (ARR) of 4.89%  with pravastatin therapy was reported for KIF6 p.Trp719Arg carriers, versus an ARR of 1.39%  for noncarriers. In WOSCOPS, ARR for KIF6 p.Trp719Arg carriers was 5.49% , versus 0.09%  for noncarriersKIF6 p.Trp719Arg carriers  versus noncarriers KIF6 p.Trp719Arg carriers with prior vascular disease , but not in noncarriers with prior vascular disease KIF6 p.Trp719Arg carriers , but not in noncarriers  (only adjusted data presented)With regards to statin therapy, all studies found thatJournal of the American College of Cardiology, Stewart and colleagues (2009) presented data that were contradictory to some of the findings published in association with Celera  Corp. The authors analyzed data from the Ottawa Heart Genomics Study (OHGS) and the Wellcome Trust Case Control Consortium (WTCCC) using the more established 9p21.3 variant as a positive control for association with MI risk/CAD, and found no association between the KIF6 p.Trp719Arg allele and CAD. They also analyzed results from the WHS, CARE, and WOSCOPS studies and felt that the data may support an association between the KIF6 p.Trp719Arg allele and MI, but not CAD. These authors did not investigate the association between the KIF6 p.Trp719Arg allele and benefit from statin therapyIn a letter to the editor of the Clinical Utility: Net benefit of test in improving health outcomes.KIF6 p.Trp719Arg testing on health outcomes were identified.No studies that investigated the impact of  KIF6 p.Trp719Arg testing were identified:Two ongoing clinical studies related toKIF6 Risk Offers Better Adherence to Statins (AKROBATS) (NCT01068834)KIF6 carrier status to study patients will improve adherence to statin therapy. The study is sponsored by Medco Health Solutions Inc. in collaboration with Celera Corp., and is scheduled to be completed in June 2011.Additional Genetic Risk Stratification to Identify Individuals for Early Statin Therapy (1RC1HL099634-01)Imprecision - despite statistically significant results, confidence intervals are wide, suggesting imprecision.Patient populations - in most studies, individuals who are homozygous for the p.Trp719Arg variants are combined with heterozygous individuals to create the "carrier" population for analysis. While this is likely to increase the power of the comparisons, it may not be appropriate given that the inheritance pattern has not been clearly established. Indeed, in all of the association studies, homozygotes were not statistically different from controls Conflict of interest - most of the published studies, and all that show a significant association between KIF6 p.Trp719Arg and CAD and/or statin response, were authored by individuals associated with Celera Corp., the parent company of Berkeley HeartLab, thereby creating the potential for conflict of interest.KIF6 p.Trp719Arg and CAD and/or statin response. While the results with respect to statin therapy appear consistent between studies, all studies are retrospective in nature which limits the applicability of the results. Finally, the lack of any studies specifically addressing the clinical utility of this information on patient outcomes is a serious deficiency in the body of evidence. Comparative studies that specifically use genotype information to stratify patients into treatment streams are needed and ideally these studies would be prospective in nature and have sufficient follow-up to determine that outcomes are indeed different in different genotype groups. Therefore, there is currently insufficient evidence to determine the utility of routine testing in patient care.The current body of evidence shows imprecise, and in some cases, conflicting results. In addition, there is lack of clarity regarding the appropriate patient populationsand the inheritance pattern of the proposed association between ClinicalTrials.gov:KIF6RePORTER (Research Portfolio Online Reporting Tool Expenditures and Results):KIF6Last updated: October 19, 2010The authors would like to acknowledge the contributions of the Hayes Genetic Test Evaluation team, particularly Lisa Spock, Linnie Wieselquist and Charlotte Kuo-Benitez.Funding for the Health Technology Assessment that informed this work was provided by Hayes, Incorporated. Funding to create this Knol was provided by the Centers for Disease Control and Prevention under Contract No. 200-2009-F-32675.This funding was provided through the Genetic Alliance.  The authors are employees at Hayes, Inc., an independent health technology research and consulting company. None of the employees at this company has any financial or personal interest in any of the technologies reviewed by Hayes, Inc.. No input on report content or conclusions is permitted by manufacturers. Although the CDC funded the work to produce this article, the content is based entirely on Hayes, Inc.'s own analysis and there was no input from the CDC.


MUTYH gene have been reported to be associated with increased risk of developing colorectal cancer. In this study, we confirmed this association using original data on 928 colorectal cancer cases and 845 healthy controls from Scotland. We then conducted a meta-analysis from published data on the association between mutations at MUTYH and colorectal cancer risk. We show for the first time a small but significant mono-allelic effect with a genotype relative risk (GRR) of 1.27 : 1.01–1.61), and confirm and give a more precise estimate of the strong bi-allelic effect with an estimated GRR of 117 (95% CI: 74–184). This study underscores the need for large sample sizes in order to identify small gene effects when the disease allele frequency is low.Mutations in the  MUTYH, which together with OGG1 and MTH1 is a key component of the base-excision repair (BER) pathway. The main function of the BER pathway is to repair DNA oxidative damage caused by aerobic metabolism. Hence, MUTYH has raised much interest among those trying to unravel the genetic contribution to cancer risk  and 1845 controls in Scotland. MUTYH homozygous mutation carriers had a 90-fold excess risk, whereas heterozygous carriers had no significant increased risk compared to wild-type homozygous on the overall cohort  and PUBMED (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) for the relevant literature references. We found 55 studies , but only eight studies contained data that met our inclusion criteria and thus were relevant to this meta-analysis. These criteria were that patients had confirmed colorectal adenocarcinoma (i.e. we excluded studies only based on the multiple adenoma phenotype), and that the study reported genotype data for cases and controls , those with one defect as WM and those with no detected defect as WW. In total, we assembled data on 13 449 people: 7273 colorectal cancer patients and 6176 population-based controls.In order to identify all relevant studies for the meta-analysis of the effect of controls . Table 3MUTYH with colorectal cancer was tested by means of a standard χ2 test with 2 degrees of freedom, and genotype relative risks (GRRs) were estimated as described previously . For the recessive model with zero cell counts, we estimated exact confidence intervals (CIs) using Fisher's exact test.P<0.05), and the results were suggestive of an association with the MUTYH gene as a whole (P=0.09). Combining these data with our previously published data revealed a highly significant association with MUTYH (P=0.0006). However, a heterozygous effect was not detected (P⩽0.0004) under the three genetic models considered. We did not find significant heterogeneity between studies under any of the models considered. The value of I2, which quantifies the level of heterogeneity on a continuous scale , whereas the overall heterozygous effect was almost significant (P=0.09).Next, we tested whether there was a homozygous (MM) and heterozygous (WM) gene effect . The oveMUTYH variant frequency was 0.34%. Similarly, data set 2 gave GRR for the MM equal to 207 (95% CI: 109–415) and 1.47 (95% CI: 1.05–2.12) for WM. Both data sets showed a highly significant (P<0.001) homozygous effect and a heterozygous effect of borderline statistical significance (P<0.05).To address the possibility that methodological differences across studies may have influenced these results, we pooled all available data described in Finally, we tested whether there were differences in the effect of Y165C and G382D mutations . EstimatMUTYH gene and colorectal cancer risk is valid. Bi-allelic inactivation of the gene conferred a very large increase in risk (GRR=117) supporting its causal role in colorectal cancer, whereas mono-allelic inativation of the gene conferred a moderate increase in risk (GRR=1.3). Confidence intervals for the estimate of the risk associated with germline bi-allelic defects were wide, even though the total sample size was over 13 000 individuals. This effect might be substantially overestimated if the proportion of compound heterozygotes in the control sample was equal to the case sample (i.e. 1/46), but we believe this is highly unlikely as we resequenced 32 (i.e. two-third) of our heterozygote controls and did not find any compound heterozygotes.This study confirms, in a combined data set of over 7000 cases across several study populations, that the association between variants in the Similarly, mono-allelic defects are of borderline statistical significance. This underscores the important role of meta-analyses of large data sets from well-conducted studies to properly interpret these relationships. It is difficult to assess whether differences in the screening method employed by different studies might explain the small heterozygous effect , but additional support from other studies (Our study confirms and provides more precise estimates for the homozygous effect and strengthens the evidence for a weak heterozygous effect. The study underscores some of the difficulties of studying the role of low-frequency variants of small effect and emphasises the need for international collaboration to achieve the very large sample sizes required to identify these variants and quantify their effects.


There is no clear consensus on the better therapy [radiofrequency ablation (RFA) versus hepatic resection (HR)] for small hepatocellular carcinoma (HCC) eligible for surgical treatments. This study is a meta-analysis of the available evidence.Systematic review and meta-analysis of trials comparing RFA with HR for small HCC published from 1997 to 2009 in PubMed and Medline. Pooled odds ratios (OR) with 95% confidence intervals (95% CI) were calculated using either the fixed effects model or random effects model.One randomized controlled trial, and 9 nonrandomized controlled trials studies were included in this analysis. These studies included a total of 1411 patients: 744 treated with RFA and 667 treated with HR. The overall survival was significantly higher in patients treated with HR than in those treated with RFA at 3 years , and at 5 year . RFA has a higher rates of local intrahepatic recurrence compared to HR . In the HR group the 1, 3, and 5 years disease -free survival rates were significantly better than in the HR-treated patients . The postoperative morbidity was higher with HR , but no significant differences were found concerning mortality. For tumors ≤ 3 cm HR did not differ significantly from RFA for survival, as reported in three NRCTs .HR was superior to RFA in the treatment of patients with small HCC eligible for surgical treatments, particularly for tumors > 3 cm. However, the findings have to be carefully interpreted due to the lower level of evidence. Hepatocellular carcinoma (HCC) is the fifth most common cancers in the world, with an estimated 500,000 deaths per year . AdvanceAmong these therapies, RFA is a promising and recently developed ablation technique. It induces deep thermal injury in hepatic tissue while sparing the normal parenchyma. Its basic principle includes generation of high-frequency alternating current which causes ionic agitation and conversion to heat, with subsequent evaporation of intracellular water which leads to irreversible cellular changes, including intracellular protein denaturation, melting of membrane lipid bilayers, and coagulative necrosis of individual tumor cells. RFA was recommended as the best treatment option for patients with early stage HCC who are not suitable for resection or transplantation in addition to PEI in the 2005 practice guidelines issued by the American Association for the Study of Liver Diseases . RecentlElectronic searches were performed of the PubMed and Medline from January 1997 to November 2009. The following Mesh search headings were used: "radiofrequency ablation," "hepatocellular carcinoma". Reference lists of all retrieved articles were manual searched for additional studies. No language restrictions were made.Two reviewers  independently extracted the following parameters from each study: 1) first author and year of publication; 2) number of patients, patients characteristics, study design; and lastly, 3) treatment outcome. All relevant text, tables and figures were reviewed for data extraction. Discrepancies between the two reviewers were resolved by discussion and consensus.For inclusion in the meta-analysis, a study had to fulfil the following criteria: 1) compare the initial therapy effects of RFA and HR for the treatment of small HCC, regardless of the etiology of liver disease, cirrhotics status, or differences in viral hepatitis; 2) report on at least one of the outcome measures mentioned below; 3) clearly document indications for RFA and HR; 4) In dual (or multiple) studies were reported by the same institution and/or authors, either the one of higher quality or the most recent publication was included in the analysis. The primary endpoints were overall survival at 1, 3, and 5 years, and local recurrence. The secondary endpoints were disease-free survival at 1, 3, and 5 years, morbidity, and mortality.Small HCC are defined according to the criteria developed by Yao et al.  from theAbstracts, letters, editorials and expert opinions, reviews without original data, case reports and studies lacking control groups were excluded. The following studies were also excluded: 1) those dealing with unresectable HCC or HCC recurrence after hepatectomy; 2) those with no clearly reported outcomes of interest; 3) those evaluating patients with cholangiocellular carcinomas or liver metastases.2. I2 < 25% and I2 > 50% reflect small and large inconsistency, respectively. P < 0.05 was considered significant.The meta-analysis was performed using the Review Manager (RevMan) software, version 4.2.7. All analyses were performed on dichotomous outcomes. Pooled odds ratios (OR) with 95% confidence intervals (95% CI) were calculated using either the fixed effects model or random effects model. Heterogeneity was evaluated by χ2 and IAfter initial screening, 19 potentially relevant clinical trials of HCC were identified. Of these, three trials did not separately analyze the results of RFA from the other local therapies -14, two There was no significant difference in overall survival between the two groups at 1 year , and at 2 years , whereas the difference was significant and favourable to HR group at 3 years   , but no differences were found between the two groups with respect to postoperative mortality . There were 1 death reported in the RFA group, and 6 in the HR group, giving a mean mortality rate of 0.1 per cent and 0.8 per cent, respectively  who were randomized for RFA converted to HR .High rate of intrhepatic recurrence after ablation therapies and/or surgical resection is the main cause of late death of patients with HCC. In current study, local recurrence was found to be more frequent after RFA than after HR. Local recurrences after RFA may be attributable to insufficient ablation of the primary tumor and/or the presence of tumor venous invasion in the adjacent liver. Surgical resection could remove the primary tumor and venous tumor thrombi . This maAs regards the distant recurrence, no differences were found between the two groups. This fnding is reasonable because the occurrence of distant recurrence is correlated with the host factors and initial tumor factors , and theAlthough the overall rate of postoperative morbidity was 32.5% in patients treated with HR, which is significantly higher than in those treated with RFA. However, these rates do not translate into an increase in mortality.The results of this meta-analysis should be interpreted with caution for several reasons. First, the majority of data in the present study comes from NRCTs, and the overall level of clinical evidence is low. Second, there are important heterogeneity between two groups. This could be due to a bias in patient selection in several series in terms of patient demographics and tumor characteristics. Patients undergoing RFA had a few less favourable characteristics such as old age, multiple nodules, severe chronic liver disease, high serum level of alpha-fetoprotein and aspartate aminotransferase level ,25,27,28Third, RFA can be performed by percutaneous, laparoscopic or open approaches. Laparoscopic and open approaches increase the chance of detection of unknown intrahepatic and extrahepatic tumors because they allow complete abdominal exploration and intraoperative ultrasound assessment. The additional advantages of open and laparoscopic approaches are the accurate placement of electrodes and the possible treatment of tumors in percutaneously inaccessible areas of the liver and tumors in close proximity to or invading the adjacent organs . On the Vascular invasion is a well-established prognostic indicator of HCC. Considering that vascular invasion was less frequent in tumors ≤ 3 cm , this maHR was superior to RFA in the treatment of patients with small HCC eligible for surgical treatments, particularly for tumors > 3 cm. However, the findings have to be carefully interpreted due to the lower level of evidence. Further RCT are needed to define the exact value of HR and RFA for small HCC, even for tumors ≤ 3 cm.The authors declare that they have no competing interests.YZhou participated in the design and coordination of the study, carried out the critical appraisal of studies and wrote the manuscript. BL, DX, ZY and developed the literature search, carried out the extraction of data, assisted in the critical appraisal of included studies and assisted in writing up. YZhao and FX carried out the statistical analysis of studies. JY interpreted data, corrected and approve the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/10/78/prepub


CRP genetic variant +1444C>T (rs1130864) and CHD risk in the largest study to date of this association.It is unclear whether C-reactive protein (CRP) is causally related to coronary heart disease (CHD). Genetic variants that are known to be associated with CRP levels can be used to provide causal inference of the effect of CRP on CHD. Our objective was to examine the association between CRP genetic variant +1444C>T (rs1130864) with CRP levels and with CHD in five studies and then pooled these analyses . CRP was associated with potential confounding factors  whereas genotype (rs1130864) was not associated with these confounders. The pooled odds ratio of CHD per doubling of circulating CRP level after adjustment for age and sex was 1.13 , and after further adjustment for confounding factors it was 1.07 . Genotype (rs1130864) was associated with circulating CRP; the pooled ratio of geometric means of CRP level among individuals with the TT genotype compared to those with the CT/CC genotype was 1.21  and the pooled ratio of geometric means of CRP level per additional T allele was 1.14 , with no strong evidence in either analyses of between study heterogeneity . There was no association of genotype (rs1130864) with CHD: pooled odds ratio 1.01  comparing individuals with TT genotype to those with CT/CC genotype and 0.96  per additional T allele . An instrumental variables analysis (in which the proportion of CRP levels explained by rs1130864 was related to CHD) suggested that circulating CRP was not associated with CHD: the odds ratio for a doubling of CRP level was 1.04 .We estimated the association of We found no association of a genetic variant, which is known to be related to CRP levels, (rs1130864) and having CHD. These findings do not support a causal association between circulating CRP and CHD risk, but very large, extended, genetic association studies would be required to rule this out. It remains unclear as to whether C-reactive protein (CRP) is causally related to coronary heart disease (CHD). Higher levels of CRP are associated with known risk factors for CHD, and these might confound the purported causal link between CRP and CHD.It has been suggested that the exploitation of the principles of Mendelian inheritance can be used to determine unconfounded and unbiased estimates of associations between non-genetic risk factors and disease outcomes,CRP gene to be unrelated to prevalent and incident CHD events, despite these variants being associated with CRP levels.This approach was used by Casas et al.CRP with CHD risk, despite associations of these haplotypes with CRP levels.CRP there were no associations with CHD events except in one sub-group analysis: AA genotype of the triallelic SNP rs3091244 was associated with prevalent coronary heart disease in non-Hispanic white individuals.CRP with cardiovascular disease events.In a recent prospective nested case control study there were no associations between four out of five common haplotypes in CRP with CHD may be beneficial in its own right.CRP with cardiovascular endpoints, but suggested that if it could be established that variants in CRP were robustly associated with CHD events then CRP gene profiling could have clinical utility in disease prediction.In addition to its importance in understanding the causal role of CRP with CHD, it has also been suggested that determining the association of variation in CRP to provide causal inference about the effect of CRP on CHD and to explore whether there is likely to be potential for using CRP gene profiling in the prediction of CHD. Ours is the largest analyses to date to examine the association of variation in any CRP SNP or haplotype with CHD (we examine association with one SNP rs1130864); our analysis includes 18,637 participants with 4,610 CHD cases. The companion paper uses data from one of the 5 studies included here to examine the association of genetic variation in CRP with carotid intima media thickness.CRP SNP rs1130864 with CHD then the companion paper will contribute to understanding whether this association is due to an association of genotype with atherogenesis, plaque rupture or both.The aim of the present study, and a companion paper,Data from the British Women's Heart and Health Study (BWHHS),CRP genotype with CHD, and the association of genotype with later outcomes cannot be explained by reverse causation or confounding, we have used a combined outcome of prevalent CHD (i.e. cases were identified at the same time that CRP was measured and blood samples for DNA were extracted) and incident CHD (i.e. cases occurred after CRP assessment and DNA extraction) for all of our main analyses. However, we also checked that associations were similar for prevalent cases only and for incident cases only.Since our main analyses are concerned with the association of In the BWHHS prevalent cases were any women with self-report of a doctor diagnosis of angina or myocardial infarction, evidence in the medical record review at baseline of either of these diagnoses, or ECG-defined ischemia. Incident CHD was defined as either death from CHD (ICD10 codes I20–I25) or evidence of new angina or myocardial infarction in medical record reviews.In Caerphilly and Speedwell prevalent CHD was defined as any man with ECG-defined ischemia or self-report of doctor diagnosed myocardial infarction or angina at baseline and incidence CHD as ECG-defined ischemia at any follow-up examination, self-report or medical record evidence of acute MI (WHO criteria), or death from CHD (ICD 9: 410–414).In the Whitehall II study, prevalent CHD was ascertained by questionnaire items on chest pain and a physician's diagnosis of a heart attack.In the HIMS, prevalent CHD was defined as questionnaire-reported coronary symptoms, bypass surgery or angioplasty, or evidence from the Western Australia Linked Data System of a non-fatal CHD event. The Western Australian Linked Data System keeps a record of all inpatient admissions and all deaths in the State.CRP with CRP and CHD in these studies. This SNP was chosen because it has been shown to be consistently related to CRP levels and is therefore a robust instrument for exploring the causal association of CRP levels with disease outcomes.CRP association with CHD,CRP SNP that had been typed in all of our new studies. Full details of how genotyping was undertaken and CRP measured for each study are provided in the supplementary material on the journal website (We assessed the association of +1444C>T SNP (rs1130864) in the 3′ untranslated region of  website .2). Information on occupation (to determine socioeconomic position), smoking and physical activity were determined from standard questionnaires.In all cohorts weight (without shoes and in light clothing) and height were assessed using standard research procedures and used to calculate body mass index . The main rationale for this was that the previously published Mendelian Randomization study Linear and logistic regressions were performed to examine the associations of potential confounding factors with CRP concentration and genotype. Linear regression was used to assess the association between genotype and log CRP, which is presented as the ratio of geometric mean comparing those who were TT homozygotes for rs1130864 compared to C allele carriers . Logistic regression was used to assess the association between log CRP and any  CHD, which is presented as the age adjusted odds ratio per doubling of CRP.2 measure that describes the percentage of total variation in the pooled estimate that is due to between study heterogeneity.Random effects meta-analyses were used to pool results from individual studies. Each individual cohort reported in the meta-analysis undertaken by Casas et al.Additional information on statistical analyses, including the instrumental variables analyses, and strengths and limitations of this method, are provided in the supplementary material on the journal website .Our analyses included 18,637 participants, of whom 4,610 were CHD cases. Across all studies 48% of participants were homozygous for the C allele, 43% were heterozygotes and 9% were homozygous for the T allele; the T allele frequency was 0.31 and the overall HWE test result . None of the percentage of participants who were male in each study, mean age of study participants, the percentage of cases that were incident in each study or the percentage of cases that were hard CHD outcomes (acute MI or death from CHD) explained between study heterogeneity in either the age adjusted or the full confounder adjusted meta-analyses .2 = 0%, p = 0.90). The pooled (for our 5 in-house studies only) per T allele ratio of geometric means was 1.13 , with no detectable between-study heterogeneity . Despite the robust associations of rs1130864 with circulating CRP this variant explained only a small proportion of its variation: within BWHHS (women only) it explained 0.8%; within Caerphilly (men only) 0.4%; within Speedwell (men only) 0.6%; within Whitehall II (73% men) 0.4% and within HIMS (men only) 0.7%.Genotype (rs1130864) was associated with circulating CRP in all cohorts , with th2 = 7.5%, p = 0.62). The pooled (for our 5 in-house studies only) per T allele odds ratio of CHD was 0.96 , with no detectable between-study heterogeneity in this analysis .There was no strong evidence of an association between rs1130864 and CHD in any of the studies.  = 0.83), justifying our assumption of no correlation in our use of Fieller's theorem for estimating the confidence interval for the instrumental variables analysis  or as measured by the correlation coefficient of −0.089 , we present evidence that higher circulating CRP level is unlikely to be an important causal risk factor for CHD. We found no association of a genetic variant (rs1130864) in CRP with CHD events, despite this variant being consistently associated with circulating CRP. Using rs1130864 to explore the causal association of CRP levels with CHD risk is valid since individuals who are homozygous for the T allele (TT genotype) will have experienced on average higher levels of circulating CRP over their lifetime than other individuals (CT or CC genotype),In this study of 4,610 cases (the study with the largest numbers to date to examine the association of variation in The assumptions underlying the Mendelian randomization approach is that the genetic variant is associated with the modifiable risk factor (circulating CRP levels in this example) and that it is not related to the outcome of interest (CHD) other than through its association with the modifiable risk factor (i.e. there are no confounding factors relating genotype to CHD and genotype is not related to CHD through other pathways).CRP genotype will not be related to socioeconomic and behavioural confounding factors that tend to distort observational epidemiological studies of this association,There are strong theoretical bases for believing that CRP that is associated with circulating concentrations of CRP and thought to be functional.CRP is in LD with rs1130864 and provides another pathway to CHD. To explain our null result in the context of circulating CRP being truly causally related to CHD this alternative pathway would have to result in a decrease in CHD risk by a magnitude that exactly reversed the posited causal influence of circulating CRP on CHD risk. However, rs1130864 exhibits no major LD (correlation R2>0.2) with SNPs in any other gene in its genomic region, except with a nearby gene (ENSG00000196401) identified in Ensembl and HapMap R2 = 0.8, but not shown in Entrez. For lower levels of LD (i.e. with R2< = 0.2), the effect of any confounding gene on CHD would have to be so large as to be implausible (no such gene has been found in any CHD genome wide studies to date The variant that we have used in this study is in close linkage disequilibrium (LD) with variation within a transcription factor binding site located 5′ of CRP and we would have had greater statistical power for our instrumental variables analyses had we constructed haplotypes using several SNPs as in one of our previous papers.CRP are not associated with CHD in the way predicted by their association with CRP levels  might limit the Mendelian randomization process. The extent to which this occurs with modest effects such as the differences in CRP level by genotype is unclear.For our main analyses we combined incident and prevalent cases. In conventional observational epidemiology incident cases are important for making causal inference and minimising any bias due to reverse causality. However, in genetic association studies reverse causality is not possible and survivor bias very unlikely.There was important heterogeneity between individual studies in the association of circulating CRP with CHD that was not explained by differences in the distribution of sex, age or the proportion of incident cases between studies. Adjustment for potential confounding factors reduced heterogeneity but some remained even in the confounder adjusted analyses, suggesting that residual confounding in some studies might explain this heterogeneity. Since our Mendelian randomization study relates the proportion of circulating CRP that is explained by rs1130864 to CHD risk, and there was no between study heterogeneity in the association of this genetic variant with CRP levels or with CHD, our Mendelian randomization results are unaffected by between study heterogeneity in the observational association of CRP with CHD.CRP genotype and CHD, the level of precision is in fact spurious since it does not fully account for the relatively small proportion of total variation in CPR accounted for by genotype and does not include uncertainty from all analyses.Our instrumental variables analysis uses the proportion of the variation in CRP that is explained by rs1130864 to provide an estimate of causal effect that is not biased by confounding, reverse causality or attenuation by errors. However, the advantage of this approach of being less biased than a conventional multivariable regression analysis comes at the cost of reduced precision. Although the point estimate of the odds ratio per doubling of CRP from the instrumental variables analysis was virtually null (1.04), the 95% confidence interval  includes the observational association, and the association found in the most recent meta-analysis of observational studies.The fact that rs1130864 explains less than 1% of the variation in CRP within each of our studies, whilst affecting statistical precision is unlikely to result in bias. As noted previously,For example, blood pressure lowering therapies explain ∼2% of the variation in blood pressure, and in participants who are randomised to either active blood pressure lowering therapy or control in randomised controlled trials there will be many other environmental and genetic factors that influence variation in blood pressure. Nonetheless, an adequately powered randomised trial of the effect of blood pressure medication on stroke (or other cardiovascular outcomes) is, rightly, accepted as unbiased evidence of the causal effect of blood pressure on stroke risk.FTO  is associated with a wide range of vascular and metabolic outcomes, including fasting glucose, insulin and lipids, with magnitudes of association that are the same as would be predicted from the association of FTO with BMI and of BMI with these outcomes in observational studies and trials of weight reduction.With respect to other Mendelian randomization studies, single SNPs that have been shown to be robustly associated with low density lipoprotein cholesterol (LDLc), and that explain less than 1% of the variation in circulating LDLc, are robustly associated with CHD, with the magnitude of this association being somewhat larger than that predicted from the causal randomised controlled trial evidence relating statins (which reduce LDLc) to CHD.CRP to explore the causal association of CRP levels with continuously measured vascular and metabolic traits and which suggest that CRP is not causally related to blood pressure, metabolic syndrome components or carotid intima media thickness.CRP gene to be unrelated to prevalent and incident CHD events, despite these variants being associated with CRP levels,CRP or CHD, in general subgroup analyses fail to replicate and should be treated with caution until replicated in other independent studies. Furthermore, none of the large genome wide association studies of CHD have found variation in CRP to be robustly associated with CHD.CRP to CHD–there does not appear to be any strong evidence for an association of CRP with CHD events, despite its association with CRP levels. These findings together would suggest that variation in circulating CRP level is not importantly causally related to CHD risk and that genetic profiling of CRP is unlikely to be useful in the prediction of CHD. However, we acknowledge that a very large genetic (Mendelian randomization) study is required to definitively demonstrate whether there is no causal association of CRP with CHD. A newly established collaboration that will include at least 30,000 CHD events will over the coming years be able to provide this definitive answer.Our Mendelian randomization findings are consistent with other studies that have used variants in CRP gene with serum CRP and carotid intima-media thickness (CIMT) in one of the studies included in this paper .CRP as instrumental variables that CRP concentration is causally related to CIMT.CRP with CIMT,In a companion paper we examined the association of 3 tag SNPs  in the Whilst our findings, together with those of several other studies described above, suggest that circulating levels of CRP are not importantly causally related to the development of atherosclerosis or CHD, an effect of CRP on case-fatality in those with CHD, is possible. Such an effect is implicated by findings from rodent models of beneficial effects of post-myocardial infarction CRP lowering,CRP with CIMT and CHD,Our findings, together with those from a number of other studies examining the association of genetic variation in Data Access: DAL had full access to all of the data from the BWHHS, Caerphilly and Speedwell studies used in this paper; MK and MGM had full access to all of the data from the Whitehall II study and NW and LJP had full access to all of the data from HIMS. These individuals take responsibility for the integrity of the data and the accuracy of the data analysis.Text S1(0.04 MB DOC)Click here for additional data file.


We aimed at assessing the degree of measurement error in essential fatty acid intakes from a food frequency questionnaire and the impact of correcting for such an error on precision and bias of odds ratios in logistic models. To assess these impacts, and for illustrative purposes, alternative approaches and methods were used with the binary outcome of cognitive decline in verbal fluency.Using the Atherosclerosis Risk in Communities (ARIC) study, we conducted a sensitivity analysis. The error-prone exposure – visit 1 fatty acid intake (1987–89) – was available for 7,814 subjects 50 years or older at baseline with complete data on cognitive decline between visits 2 (1990–92) and 4 (1996–98). Our binary outcome of interest was clinically significant decline in verbal fluency. Point estimates and 95% confidence intervals were compared between naïve and measurement-error adjusted odds ratios of decline with every SD increase in fatty acid intake as % of energy. Two approaches were explored for adjustment: (A) External validation against biomarkers (plasma fatty acids in cholesteryl esters and phospholipids) and (B) Internal repeat measurements at visits 2 and 3. The main difference between the two is that Approach B makes a stronger assumption regarding lack of error correlations in the structural model. Additionally, we compared results from regression calibration (RCAL) to those from simulation extrapolation (SIMEX). Finally, using structural equations modeling, we estimated attenuation factors associated with each dietary exposure to assess degree of measurement error in a bivariate scenario for regression calibration of logistic regression model.Attenuation factors for Approach A were smaller than B, suggesting a larger amount of measurement error in the dietary exposure. Replicate measures (Approach B) unlike concentration biomarkers (Approach A) may lead to imprecise odds ratios due to larger standard errors. Using SIMEX rather than RCAL models tends to preserve precision of odds ratios. We found in many cases that bias in naïve odds ratios was towards the null. RCAL tended to correct for a larger amount of effect bias than SIMEX, particularly for Approach A. R) may also affect the FFQ-based assessments (Q), which include over or under-reporting biases of subjects with certain socio-demographic or health-related characteristics. This problem would make it impossible to presume independent random errors in the two methods, which in turn leads to over-estimation of the correlation between the reference method and the FFQ [Food frequency questionnaires (FFQ) have been historically used in large epidemiological studies to assess usual dietary intake of specific nutrients and food groups. They have been repeatedly validated against another dietary assessment method assumed to be more accurate such as multiple 24-hour recalls or food records of food intake -3. The p the FFQ .External validation against two biomarkers measured at visit 1 among a subset of the Atherosclerosis Risk in Communities (ARIC) study. These concentration biomarkers are the percent of each fatty acid or group of fatty acids out of total fatty acid concentration in the cholesteryl ester (M) and phospholipids (N) fractions of plasma which were previously shown to be linearly related to dietary intake as assessed by more reliable reference methods such as multiple 24-hour recalls, food records or diet history [Internal repeat measurement of the FFQ values at visits 2 and 3. While the former approach eliminates the possibility of correlated errors between test and reference method (one being a self-report and the other a biomarker), the second approach is often more readily available in many large cohort studies. In addition, for the second objective, we contrasted regression calibration with another method known as simulation extrapolation or SIMEX. Previous research indicated that dietary n-3 fatty acids were suggestive of a protective effect against cognitive aging [n-3 fatty acids on cognitive decline and to define exposure in terms of absolute value of medium chain and long chain fatty acids, as well as the ratio between n-3 and n-6 fatty acids in diet and plasma [The present study aims first at assessing the degree of measurement error in the intake of essential fatty acids using a food frequency questionnaire, by estimating attenuation factors for each exposure. Second, we studied the impact of correcting for such measurement error on bias and precision of odds ratios from a multivariate logistic regression model. For illustrative purposes, we examined the effect of dietary essential fatty acids on clinically significant cognitive decline among older adults, controlling for other potential confounders. For both objectives, two alternative approaches were used: (A)  history -8. In fave aging -13. Otheve aging -18. Howeve aging ,20. An ed plasma .Findings from our study and its overall methodology may be used in subsequent analyses to adjust for measurement error in primary regression linking intake of essential fatty acids with various disease outcomes.Q1, Q2 and Q3. For M and N, they were expressed as % of total fatty acids in the plasma fraction. A decreasing trend in reported consumption of groups 6P and 6 was noted. Similarly, the ratio of reported 3H/6H declined almost linearly over visits 1, 2 and 3. Standard deviations of Q1 are instrumental in interpreting results for logistic regression model analyses.Table M/N) and B . Our findings suggested that attenuation of bivariate effects was more pronounced for approach A with factors ranging between 0.046 for 6H and 0.426 for 3H/6H. As for Approach B, attenuation factors ranged between 0.456 for 3P and 0.655 for 3H/6H and 3/6. Further, a sensitivity analysis with assumed naïve odds ratios of two for all j variables (Loge(ORnaive) = 0.693 with SE = 0.10) was conducted to get a feel of the magnitude in attenuation in a measure of association that is commonly used in epidemiologic studies. Equations 4.1–4.4 (Methods section) were used for this purpose. In many cases, approach A yielded corrected odds ratios that were outside the reliable range (The two odds ratios differed by more than 10-folds) while approach B gave adjusted odds ratios within a reliable range. However, further sensitivity analysis with other values of the naïve estimate of the odds ratio indicated that a value between 1.0 and 1.5 gave more reliable corrected estimates compared to values of 1.5 or higher, particularly for approach A. It is worth noting that these computations are only applicable to bivariate models with one error prone variable.Table Figure bias| = 0.12); Approach A/SIMEX (|Δbias| = 0.06); Approach B/RCAL (|Δbias| = 0.08); Approach B/SIMEX (|Δbias| = 0.06). Hence, in general, RCAL corrected for more bias than SIMEX did and this was more obvious for Approach A.As an application to multivariate logistic models, we conducted further analysis using available data on cognitive decline and essential fatty acids and controlling for relevant confounders (Equations 4 and 5). Table Q2 was measured for only 657 subjects out of the 7,814 who were eligible. Comparing the two approaches, many of the unexpected results with bias through or away from the null occurred in Approach B. In addition, in terms of precision, approach A yielded an average for ratios of confidence limit ratios (CLRs) of 1.22 (1.41 for RCAL and 1.14 for SIMEX), compared to 1.62 (1.75 for RCAL and 1.48 for SIMEX) for approach B, which indicates a greater overall precision in approach A.For internal repeat measurements (Approach B), results indicated an appreciable loss in precision, given that replicate β1 = LogeOR), Figure 3H (z-score) and odds of decline in WFT corrected using both approaches A (concentration biomarkers M and N) and B (replicate measures of Q1: Q2 and/or Q3). In both cases, 6H was included as an error prone confounder and the effect of measurement error on its associated regression coefficient is also presented. It is clear that while approach A revealed an underestimation of the protective effect  < 0, assuming the true value of 1) of 3H on WFT decline by an absolute value of 0.09 between the naïve and the SIMEX coefficients, approach B had a discrepancy of no more than 0.05. The same difference in underestimation of adverse effect  > 0) is noted for 6H.To illustrate graphically the SIMEX procedure that allows for correction of the point estimate of effect (Q2 (n = 634) and Q1 . However, it is important to note that the main study population differed significantly from the sub-study with external validation data (i.e. biomarkers). In contrast, demographic and lifestyle differences were not as striking for the repeat measurements sub-study, probably due to the relative preservation of racial and ethnic diversity in this sub-group. Hence, transportability of attenuation factors from the validation sub-study is more questionable for the subgroup with available biomarker data . In addition, although loss of precision is an issue, it may also be viewed by some as yielding more conservative estimates of effect particularly if this loss is not substantial, as is the case for SIMEX. Hence, we recommend using SIMEX rather than RCAL whether the choice is to use biomarker measures results (Approach A) or replicate measures (Approach B). As expected, while RCAL gave less precise estimates, however, it corrected for a larger amount of bias in odds ratios, particularly for Approach A.Our main findings suggested that estimates were appreciably less precise for internal repeat measurements when compared to external validation with biomarkers. However, they gave more conservative results regarding the association between fatty acid groups and ratio with cognitive decline in WFT. For instance, based on biomarker/RCAL results, one could conclude that a 1 SD increase in 3H (n-3 highly unsaturated fatty acids) may reduce the odds of decline by as much as 72% or as little as 10%, with an average of 27%. While the point estimate for the repeat measures approach still indicated a protective effect of 3H, both RCAL and SIMEX analyses resulted in broad confidence intervals that crossed the null value of 1. These findings can be contrasted with the study by Duffy and colleagues , who fouThe present study is also one of the very few attempts to estimate an attenuation factor for essential fatty acids as exposures that can be used subsequently by other researchers for the purpose of correcting for measurement error in bivariate generalized linear models. The approach used was similar to previous research -25. Whil methods . Aside f methods ,28, stro methods ,30, type methods ,33, depr methods ,35, a hy methods ,37 and C methods .λj was 0.2, the sample size, calculated by assuming that λj is equal to 0.4, should be multiplied by 0.42/0.22 = 4 to achieve the same nominal power [One major implication to measurement error, as stated earlier, is loss of statistical power to detect an exposure-disease association. In fact, the sample size required to detect a specific odds ratio (e.g. OR = 2) is inflated proportionally to the inverse squared attenuation factor. For instance, if the true al power . It is wSome of the main limitations of this study include the lack of a reference method that is known to be more reliable than FFQs in the ARIC study . However, because of correlated errors between self-report methods, the use of biomarkers has often been cited as a more adequate means to assess the extent of measurement error in a test instrument. Another drawback is the fact that plasma levels of fatty acids in both fractions studied constitute a short-term measure of intake although they have been shown to correlate well with long-term intake . In addiFuture endeavors to correct for error should make use of structural equations modeling and include as many concentration biomarkers as is available along with other self-reported or biomarker-based reference methods of dietary assessment. However, the choice of biomarkers and interpretation of their variability must be made as to account for biochemical and physiological interactions between dietary, environmental and genetic factors. Moreover, one must be cautious of coupled errors between biological markers and must take into account these correlations when specifying the structural model. Finally, because structural equations modeling makes a strong assumption about joint multivariate normality, often not present, it is crucial for future studies to use newly developed methodologies which appear to be more flexible in many ways .ARIC is a prospective cohort study which aimed at investigating the etiology of atherosclerosis and its clinical sequelae and the longitudinal impact of variation in cardiovascular risk factors, medical care, and disease by race, sex, place, and time. In each of four US communities – Forsyth County (NC), Jackson (MS), suburbs of Minneapolis (MN), and Washington County (MD) – 4,000 adults aged 45–64 years were examined four times, three years apart (visits 1 through 4). Three out of the four cohorts represented the ethnic mix of their communities, while at Jackson, MS, only African American residents were recruited . Out of Measures of cognitive functioning were made for visits 2 and 4 of the ARIC study. In our present study, we focus on decline in Word Fluency Test (WFT). This test requires subjects to record as many words as possible using the initial letters F, A and S and to list these words, the subject is given only 60 seconds per letter. The total score corresponds to the total number of words generated during these three trials. The test is particularly sensitive to linguistic impairment ,48 and e2-X1)-(M2-M1))/S.D., where X1 is the individual's score at baseline, X2 the individual's score at follow-up, M1 and M2 are the group mean pretest and follow-up scores respectively, and S.D. the observed standard deviation of the difference scores. Scoring below an RCI of -1.645 was regarded as a "statistically reliable" deterioration in the test scores.Cutoff points were determined for decline in cognitive status WFT test using the Reliable Change Index (RCI) method in order to correct for measurement error and practice effects . RCI is Usual dietary intake was estimated from an interviewer-administered 61-item semi-quantitative food frequency questionnaire (FFQ) previously developed and validated by W. Willet and colleagues against multiple food records among a sub-sample of the Nurse's Health Study cohort .(3P) n-3 C18 polyunsaturated fatty acids: 18:3+18:4n-3 (6P) n-6 C18 polyunsaturated fatty acids: 18:2+18:3n-6 (3H) n-3 C20 and C22 highly unsaturated fatty acids (HUFAs): 20:5+22:5+22:6n-3 and (6H) n-6 HUFAs: 20:3+20:4+22:4+22:5n-6. Sums of fatty acid intake as percent of energy included (3) = (3P)+(3H) and (6) = (6P)+(6H). Ratios of interest included (3P)/(6P), (3H)/(6H) and (3P+3H)/(6P+6H) also denoted as 3/6. In multivariate models, all exposure variables were standardized by subtracting each observation from the variable mean and dividing the difference by the standard deviation. Adjustment was made for the other fatty acid variables when appropriate, and total energy intake was considered as a potential confounder to emulate a multivariate nutrient density model [In our study, dietary intake of essential fatty acids and their elongated and desaturated products were expressed as percent of total energy intake and grouped under four main categories, as suggested by Lands and colleagues ,54: (3P)ty model . While oTwelve-hour fasting blood was collected according to the ARIC study wide protocol. The Minneapolis field center conducted fatty acid analysis in plasma phospholipid and cholesteryl ester fractions for visit 1 blood specimens (1987–89) among the white segment of the study population in that center. The procedure is described in detail elsewhere . The ideDietary intake was assessed among the surviving ARIC sample at visit 3 (1992–94), using the same FFQ that was administered at baseline. At visit 2 (i.e. 1990–92), a sub-sample of ARIC  was asked to repeat the FFQ, unlike visits 1 and 3 in which the whole ARIC sample was covered. As stated earlier, of our eligible subset with baseline data on exposure and complete outcome data , 657 had data on visit 2 exposure, 7,482 had complete data at visit 3, while 634 had both.ε4 carrier status), behavioral , nutritional . All these were previously shown to be independently predictive of the outcome and associated with our exposure. The distributions of these covariates within each sub-sample considered are presented in Table Most covariates considered as potential confounders were measured at visits 1 or 2. These included sociodemographics , genetic (Apo E Bivariate scenario: estimation of attenuation factors(1) . In this part, each essential fatty acid using the two alternative approaches and a structural equations modeling technique. Multivariate scenario: regression calibration and Simulation Extrapolation(2) . In this part, the association between essential fatty acid intake and clinically significant decline in WFT as an outcome was studied and adjusted for measurement error in exposures. We examined changes in 95% CI and point estimates after correction for error using alternative approaches and methods.(Eq. 1 and 2).Estimating the attenuation factor associated with the effect of each exposure variable or covariate on an outcome constitutes the first step for measurement error adjustment using regression calibration. In our example, and for attenuation factor estimation, the structural models were considered for each of the two approaches Approach AApproach Bλj with its approximate SE, using the delta method (Eq. 3).This set of equations allowed us to estimate the attenuation factor 1) = Var(T) = 1. This is the case when all measured variables  are standardized z-scores.when Var with a response variable Y contains only one error-prone explanatory variable Qj (bivariate scenario) correction for attenuation, consists simply of dividing the regression coefficient β of that variable Qj by the attenuation factor λj (Eq. 4.2). The variance estimates for the corrected regression coefficient of effect as well as 95 percent CI are estimated using Eqs. 4.3.1, 4.3.2 and 4.4. Qij 1and biomarkers Mij and Nij are entered into the model as z-scored variables. The same applies to the repeat measures Qij 2and Qij. 3Note that Qij 1is the manifest variable denoting visit 1 dietary exposures, which are the test exposures of interest .Assuming non-differential misclassification, dietary measurement error often attenuates diet-disease relationship and thus biases the effect towards the null. The amount by which the association is biased can be estimated by the attenuation factor . AccordiβRC : (i) β21 * σ2 product is small; where σ2 = Var ; (ii) Pr(Y = 1|T) is small and f(T|Q) is normal.For logistic regression, the assumptions made are linear homoscedastic regression of T on Q with a normally distributed error term and a rare disease requirement . Subsequ(5), the attenuation factor is no longer the parameter to be used for calibration.In cases where more than one error-prone variable and several perfectly measured variables are introduced into a primary regression model For the multivariate scenario, we computed odds ratios of clinically significant decline in WFT (RCI < -1.645) with increase in each exposure by 1 SD through a multivariate logistic regression analysis. Control for confounding was accomplished using backward elimination whereby covariates that changed the estimated effect of the exposure by more than 5% were retained in the final model . The parQj ((6) [Under such a multivariate scenario, regression calibration becomes reliant on the variance-covariance matrix of error in the measurement of different error-prone variables tion (6) .7.1 through 7.5 [The detailed explanations of each moment is laid out in equations ough 7.5 , and tabExample: FFQ visit 1 z-score for fatty acid exposures (Q1) are error-prone variables and have at most two replicates: 1 ≤ k ≤ 3 for each observation. Z (e.g. age) is available for all observations and is not error-prone.1, Q2/Q3} or {Q1, M/N}.k replicates for each individual i. W = {QAlternatively, one can use simulation extrapolation (SIMEX) which also relies on the method of moments with an estimate of s follows:Step 1 : Simulation step:• Create additional datasets with increasingly larger amounts of measurement error, after estimating error variance for each error-prone variable (θ)θ = {0.5,1.0,1.5,2.0}(1 + (Eq. 6–7.5)• Regression coefficients estimated using method of moments Step 2: Extrapolation step:θ = {0.5,1.0,1.5,2.0}on the x-axis, the coefficient βT, SIMEX is estimated by extrapolating backwards to a value of θ = -1, where covariate Q becomes error-free,After plotting each of the estimated coefficients with Step 3: Variance and SE of the regression coefficients for SIMEX point estimates are estimated either with an asymptotic or bootstrap method.The author(s) declare that they have no competing interests.MAB: Conception, literature review, methods, plan of analysis, data analysis, interpretation of results.JSK: Conception, methods, plan of analysis, literature review, revision of manuscript.JI: Methods, plan of analysis, revision of manuscript.JS: Conception, literature review, plan of analysis, revision of manuscript.GH: Provision of data, plan of analysis, revision of manuscript.The pre-publication history for this paper can be accessed here: