The planned nationwide implementation of mammography screening 2007 in Germany will increase the occurrence of mammographically detected breast abnormalities. These abnormalities are normally evaluated by minimal invasive core biopsy. To minimize false positive and false negative histological findings, quality assurance of the pathological evaluation of the biopsies is essential. Various guidelines for quality assurance in breast cancer diagnosis recommend applying the B-classification for histopathological categorization. However, to date there are only few studies that reported results about reliability and validity of B-classification. Therefore, objectives of our study are to determine the inter- and intraobserver variability (reliability study) and construct and predictive validity  of core biopsy evaluation of breast abnormalities. This paper describes the design and objectives of the DIOS Study.All consecutive asymptomatic and symptomatic women with breast imaging abnormalities who are referred to the University Hospital of Halle for core breast biopsy over a period of 24 months are eligible. According to the sample size calculation we need 800 women for the study. All patients in the study population underwent clinical and radiological examination. Core biopsy is performed by stereotactic-, ultrasound- or magnetic resonance (MR) guided automated gun method or vacuum assisted method. The histopathologic agreement (intra- and interobserver) of pathologists and the histopathologic validity will be evaluated. Two reference standards are implemented, a reference pathologist and in case of suspicious or malignant findings the histopathologic result of excision biopsy. Furthermore, a self administrated questionnaire which contains questions about potential risk factors of breast cancer, is sent to the participants approximately two weeks after core biopsy. This enables us to run a case-control-analysis  to investigate the predictive values of various risk factors on breast cancer risk.The analysis of reliability and validity of the histopathological evaluation of core biopsy specimens of breast abnormalities is intended to provide important information needed for a high quality in breast cancer diagnostic and for planning of treatment strategies. The number of women with mammographic abnormalities of the breast will considerably increase in Germany especially in the coming years when the mammography screening will be offered population-wide. In most of these cases a breast biopsy with subsequent histological evaluation is required. In general, a breast biopsy is recommended in patients with category IV (suspicious) or V (highly suspicious) in breast imaging reporting and data system (BI-RADS). If breast biopsy does not correlate with radiological findings, a rebiopsy is recommended.An excision biopsy is performed in case of malignant or suspicious lesions. To minimize false positive or false negative histological findings, quality assurance of the pathological evaluation of the biopsies is essential. Currently, guidelines recommend to apply a pathology classification scheme that includes five reporting categories  may be misclassified as biopsies that implicate further invasive evaluation of the breast lesion (B3-B5) . For example, a pathologist could mistakenly diagnose an apocrine atypia in lobules, ducts, or sclerosing lesions as ductal carcinoma in situ (DCIS). Secondly, lesions that implicate further invasive evaluation of the breast lesion (B3-B5) may be misclassified as benign biopsies (B1-B2) which results in a lack of further invasive diagnostic evaluation . Common causes of false-positive and negative diagnoses are listed in table  may be mThere are only few available studies on the reliability and validity of the B-classification of pathological evaluation of core biopsies among women with radiographic abnormalities as has been suggested by the EC Working Group on Breast Screening Pathology or the NHSBSP but none of this used B-classification to evaluate intraobserver reliability. Britton et al. performed 202 core biopsies (1% of women screened) . For 111Ibrahim et al. studied the role of core biopsy in the preoperative assessment of impalpable breast lesions mainly among women from the National Health Service Breast Screening Programme (NHSBSP) using an extended B-categorization . They coThe diagnostic agreement in evaluation of core biopsy specimen was investigated by Collins et al. in 2004 core biopsies . The diaIn the context of a mammography screening pilot project in Bremen, Germany, Bonk et al. investigated the agreement in B-categorization of two pathologists . They reLee et al. performed a study to investigate the predictive value of the two borderline B-categories, B3  and B4  . They foA major problem of validation studies of the B-categorization of breast biopsies originates from a lack of a reference standard of the biopsy evaluation. The most valid reference standard would be a diagnostic surgical excision biopsy. However, those women with B-categories B1-B2 most likely do not undergo any invasive workup so that the reference standard information of these women is missing and may bias validation results. Possible practical solution of this problem is the composition of an independent expert panel that can assign a final diagnosis to each patient. Alternative approach is to follow up the clinical course of each patient during a suitable predefined period, in terms of an delayed-type cross-sectional study design .There are many factors that may influence the reliability and validity of the pathological evaluation of core biopsies including age of the women, gynaecological history , prior clinical, radiological or histological findings, size and type of the lesion, the representativeness of the biopsy in relation to the imaging findings and the quality of the preparation and staining of the tissue. Therefore the primary aims of the study are to evaluate the intra- and inter-observer variability of the pathologic evaluation of core biopsies of the breast, potential determinants of a disagreement within and between pathologists, the validity  of the pathologic evaluation and the potential determinants of false-positive and false-negative findings.By its very nature, the study design additionally enables us to run a case-control-analysis . Hence we can additionally investigate the predictive values of various risk factors on breast cancer risk. Secondary objectives of the study are amongst others:- to investigate predictive values of BI-RADS for malignant findings in core biopsy and excision ,11- to investigate the influence of breast density on breast cancer risk - to reassess the hypothesis of Band et al. , relatedThe study population compromises all women who undergo image-guided core biopsy for evaluation of breast abnormality at University Hospital of Halle over a 2-year period (April 2006 to March 2008) and give informed consent for participation in the study inclusiving any follow-up evaluations related to the study. The majority of women (> 90%) undergoing a mammography-, ultrasound- or MR-guided biopsy are referred to University of Halle from private outpatient radiologists and gynaecologists because of symptomatic abnormalities of the breast or for self-referred breast cancer screening, that means for a mammography in symptomless women. Women with principal residence outside of Germany or insufficient command of German language are excluded from the study.The choice of the imaging method  and the biopsy technique (automated gun methods or vacuum assisted method) for histological work-up of suspicious breast abnormalities may depend on the imaging method that makes the lesion detectable, the size and kind of the lesion, the sensitivity and specificity which is required, patient comfort and costs ,2.Generally, before a biopsy is recommended, a complete diagnostic work-up of the suspicious breast lesion  is necessary. Therefore, all clinical and imaging data from referred patients have to be reviewed again. Further necessary clinical and imaging assessments are performed at the University of Halle.According to international guidelines, ultrasound-guided core biopsy is the method of first choice because it is easier to perform more comfortable for the patient and less time-consuming than the x-ray guided techniques ,2. VacuuUltrasound-guided core biopsy is performed at University of Halle by using a programmable automatic biopsy system . To get enough specimens for histopathology, the acquisition of at least 5 cores should be routinely attempted. Ultrasound guided vacuum biopsy  is used for small lesions. The acquisition of a large tissue volume promises an excellent diagnostic accuracy.In order to get a high quality, the procedures and the documentations are standardized. The documentation of ultrasound-guided procedures should include images of the lesion before biopsy, the needle in front of the lesion and the needle within the lesion and images after biopsy.Stereotactic vacuum-assisted breast biopsy is performed on a digital prone table  using 11 gauge or 8 gauge vacuum probes . In all cases, number of cores taken should be no less than 20 (11 gauge probe) . On the - before vacuum biopsy, two orthogonal mammograms should be available- access is chosen in a way that in case of malignancy the excision of the biopsy channel is possible - the following images are documented: scout, 15°images for targeting, two pre- and post-fire-images, two images with the cavity- after VB a control mammogram is taken- in case of microcalcifications a specimen radiograph is necessary.- in cases where the whole or a high proportion of the mammographic lesion has been removed, a small metal marker clip is deployed at the biopsy site.MR-guided vacuum biopsy is performed on Siemens MR scanners (Impact Expert and Vision) using a dedicated breast biopsy coil and a VB-device . More than 20 cores  should be acquired.During the procedure the patient is placed in prone position on the compression unit. The breast is fixed between two plates. To reidentify the lesion, the entire breast is measured before and after injection of contrast media. Based on the MR images, coordinates for access to the lesion are calculated. The pathway to the lesion is precut by a rigid and thick needle. Then, a thinner needle, called "substitute needle" is inserted into the breast to imitate the position of the biopsy needle. After a control measurement this "substitute needle" is replaced by the biopsy needle and vacuum biopsy is performed in the usual way: The representative removal is checked by scanning the entire breast again before and after injection of contrast media.Figure Subsequently results of the pathological assessment are sent to the study centre. Here the results of the assessment by the first pathologist are compared to reference pathologist. In case of disagreement a consensus conference is organized. Thereafter, the treating clinicians are informed about the diagnosis by the reference pathologist respectively the consensus diagnosis.The comparison of the primary and reference pathologists enables us to calculate the sensitivity, specificity, and predictive values of the B-categorization in the context of the construct validity. The construct reference, which has a subjective component included, obviously is not a perfect objective gold standard and there may be few misclassification errors of the reference pathologists. Therefore, the follow-up information of patients improves the reference standard. The motive to choose the pathologist at University of Münster as a reference pathologist is the following: he is an internationally well-known pathologist with a special expertise in breast pathology, is one of the most experienced pathologist of breast pathology in Germany and is chair of the German Reference Center of Breast Pathology.A "second" pathologist from the Albertinen-Pathology Hamburg, who is blinded to the evaluations of the first pathologist, evaluates the identical set of slides that the first pathologist has seen in order to assess the interobserver reliability.Finally, after at least 6 months, all pathologists will again evaluate a random sample of the histological slides blinded to their own first evaluation of the specimens. The comparison of the repeated evaluation ensures the assessment of intraobserver reliability.All pathologists receive clinical data and basic data from mammography and other imaging procedures  because this information will be used by pathologists in a routine care situation as well . For womFor all biopsies with uncertain correlation of imaging and histopathology, with uninterpretable tissue due to artefacts or with specimens containing only stroma, a repeated biopsy or excision by open biopsy is necessary. In order to discuss causes and further recommendations all cases with non-representative pathology and cases of malignancy are analysed by an interdisciplinary tumor conference consisting of a gynaecologist, radiologist and pathologist as well as other medical personnel.After histopathological evaluation, it is documented, whether the histopathological findings correlate with the imaging findings. In case of malignancy, an open biopsy is recommended. The result of excision biopsy will be used as reference standard in validation of suspicious or malign core biopsy findings.In a consensus meeting at the end of the recruitment period, discordant pathology evaluations of the first, second or reference pathologist are reviewed among an expert panel consisting of the participating pathologists, radiologists and treating clinicians. Reasons for disagreement will be assessed and analyzed.A self-administered questionnaire, which contains questions about potential risk factors of breast cancer, is sent to the participants approximately two weeks after biopsy. It is sent back via reply-paid envelope.For women who underwent an open excision shortly after the core biopsy the reference standard is the histopathological examination of the excision material. According to guidelines , women wIn addition, the treating physician outside the hospital is asked to do a mammography three years after biopsy. Depending on the core biopsy technique used at entry into the study, women also undergo an ultrasound examination of the breast (for those women with ultrasound-guided core biopsy at entry) or MR of the breast (for those women with MR-guided vacuum biopsy at entry) three years after entry into the cohort. At the end of the follow-up period, women with newly diagnosed breast lesions will be compared with the imaging findings at entry into the cohort. The follow-up of these women is meant as an outlook. Currently, the study grant does not include the financial support for the follow-up.In general, for the data collection, standard case report forms (CRF's) are used if available.The radiological CRFs are based on the BI-RADS lexicon classification form . At leasAll pathologists filling out a CRF with information to kind of benign and malign lesions, histological calcification, histological grading in case of invasive cancer, B-classification, level of safety of B-category, quality of slides, details of the evaluation, need for immunhistochemical staining. If IHC staining is performed, a separate section of the CRF is completed.The self-administered questionnaire contains questions about sociodemographic data, prior breast lesions, prior breast biopsies, prior breast cancer, breast related symptoms, prior radiotherapy of the breast, menopause status , pregnancies, familial breast cancer, hormone intake, smoking history, nutrition habits, alcohol drinking, health status, anthropometric data, comorbidity, patient satisfaction and kind/location of treatment after biopsy.For reasons of quality assurance, all participating pathologists received a training set of histological slides, provided by the reference pathologist that contains 20 cases of all kinds of breast pathologies. These slides were evaluated and a B-categorization was assigned. Thereafter, the categorization was compared with the diagnosis provided by the reference pathologist. All disagreements were discussed by participating pathologists during a kick-off meeting.In order to ensure a highly standardized data collection, all study personnel is specifically trained for the study procedures. Standard Operating Procedures (SOPs) have been written for all workflows.All paper documentation is double entered in order to minimize errors due to data entry. Visual and computerized plausibility checks are performed to detect possible data entry errors of CRF and questionnaire documentation.After selection of questionnaire- and CRF- items and planning of logistic procedures, a pretest was performed to test instruments and logistics of data collection. Slight modifications were made before recruitment of the study subjects started.The planning of the DIOS-Study was performed under consideration of the GEP  and the STARD – initiative guidelines ,18.The agreement between two pathologists who independently evaluate the core biopsy material will be calculated as observed agreement and chance-corrected agreement (kappa). To assess validity of the B-categorization of first pathologist compared to the reference standard (reference pathologist respectively result of excision histology), standard measures for diagnostic tests  are calculated.The case-control analysis includes the calculation of frequency distributions and the estimation of odds ratios (OR) by unconditional logistic regression analyses. 95% confidence intervals (CI) are calculated to evaluate the precision of the estimates.All statistical analyses will be performed with SAS, Version 9.1 .The sample size calculation was done according to the primary outcome "Agreement of two pathologists relating to B-classification". Assuming a conservative proportion of true B3-B5 of about 0.20, a kappa of 0.80 and a distance from kappa to the confidence limit of 0.05, we will need about 886 patients. If the chance-corrected agreement would be larger, smaller sample sizes would be required.The assumed proportion of expected successes of 0.20 is a conservative assumption which has been taken as a safeguard against the type-II-error. Within the NHSBSP the prevalence of malignant breast disease was 31.9% . In the For the validation study part , we assumed a high specificity of 90% and more and therefore base our sample size calculations on the precision of the estimate of the sensitivity. If we assume a 95% CI, a two-sided calculation method, an expected conservative sensitivity of 70% and a distance from the point estimate of the sensitivity to the confidence limit of 0.05, we will need about 323 women in the study. Therefore, the sample size of the reliability study will be sufficient for the validation study.According to experiences of recent years, it is expected that about 600 women per year undergo breast biopsies at the University of Halle. Therefore, over a recruitment period of 24 months, we expect about 1200 women with breast biopsies. Due to exclusion criteria and refusals to participate, we expect about 800 women to participate.According to the German GEPs, all collected data is considered to be confidential . TherefoThe study is a strictly observational study so that there are no risks to declare. All eligible women will be informed about the study and have to give an informed consent before they can be included in the study.The study was approved by the Ethics Committee of the Medical Faculty of the Martin-Luther-University Halle-Wittenberg, May 22nd, 2006.With the analysis of reliability and validity of histopathological evaluation of core biopsy specimens of breast abnormalities, the DIOS study is intended to contribute important information needed for a high quality in breast cancer diagnostic and for planning of treatment strategies. The knowledge of factors, influencing the reliability and validity of breast biopsy evaluation, are intended to use for the planned German mammography screening. The main goal of the study is to intensify the interdisciplinary breast cancer research and to improve the transfer of knowledge and results between different research divisions.AEDT Atypical Epithelial Proliferation of Ductal TypeBI-RADS Breast Imaging Reporting and Data SystemCI Confidence IntervalCRF Standard Case Report FormCK CytokeratinDCIS Ductal Carcinoma In SituDIOS Diagnosis Optimisation StudyDH Ductal HyperplasiaGEP Good Epidemiologic PracticeHE Hematoxylin and EosinIHC ImmunhistochemicalLIN Lobular Intraepithelial NeoplasiaMR Magnetic ResonanceNHSBSP National Health Service Breast Screening ProgrammeOR Odds RatioSOP Standard Operating ProceduresSMA Smooth Muscle ActinThe author(s) declare that they have no competing interests.AK: coordinates the study, participates in the statistical analyses and drafted the manuscript.PT: coordinates the study, participates in the statistical analyses and helped drafting the manuscript.AH: helped to develop the study, performs the radiological examination and core biopsies and critically revised the manuscript.HJH: helped to develop the study, performs the first histopathological evaluation and critically revised the manuscript.CT: performs the first histopathological evaluation and critically revised the manuscript.SH: performs the first histopathological evaluation and critically revised the manuscript.WB: performs the reference histopathological evaluation and critically revised the manuscript.TD: performs the reference histopathological evaluation and critically revised the manuscript.TL: performs the second histopathological evaluation and critically revised the manuscript.ASP: helps coordinate the study and helped drafting the manuscript.CT: performs the surgical treatment of women and critically revised the manuscript.TLa: performs the surgical treatment of women and critically revised the manuscript.JB: performs the first histopathological evaluation and critically revised the manuscript.AS: conceived of the study, designed major parts of the study, participates in the statistical analyses and helped drafting the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Finding the genetic causes of quantitative traits is a complex and difficult task. Classical methods for mapping quantitative trail loci (QTL) in miceuse an F2 cross between two strains with substantially different phenotype and an interval mapping method to compute confidence intervals at each position in the genome. This process requires significant resources for breeding and genotyping, and the data generated are usually only applicable to one phenotype of interest. Recently, we reported the application of a haplotype association mapping method which utilizes dense genotyping data across a diverse panel of inbred mouse strains and a marker association algorithm that is independent of any specific phenotype. As the availability of genotyping data grows in size and density, analysis of these haplotype association mapping methods should be of increasing value to the statistical genetics community.We describe a detailed comparative analysis of variations on our marker association method. In particular, we describe the use of inferred haplotypes from adjacent SNPs, parametric and nonparametric statistics, and control of multiple testing error. These results show that nonparametric methods are slightly better in the test cases we study, although the choice of test statistic may often be dependent on the specific phenotype and haplotype structure being studied. The use of multi-SNP windows to infer local haplotype structure is critical to the use of a diverse panel of inbred strains for QTL mapping. Finally, because the marginal effect of any single gene in a complex disease is often relatively small, these methods require the use of sensitive methods for controlling family-wise error. We also report our initial application of this method to phenotypes cataloged in the Mouse Phenome Database.The use of inbred strains of mice for QTL mapping has many advantages over traditional methods. However, there are also limitations in comparison to the traditional linkage analysis from F2 and RI lines. Application of these methods requires careful consideration of algorithmic choices based on both theoretical and practical factors. Our findings suggest general guidelines, though a complete evaluation of these methods can only be performed as more genetic data in complex diseases becomes available. The discovery of genes that directly affect human health is an active area of biomedical research. Although studies have been historically biased toward the role of individual genes in disease phenotypes, most complex diseases are caused by the influence of numerous genes with lesser individual effects. Current efforts in mapping quantitative trait loci (QTL) seek to unravel these complex mechanisms through the identification of one or more genetic loci that influence specific phenotypes.All QTL mapping approaches have three components in common: a population of individuals with a measurable phenotypic diversity, a measure of the genotypic diversity present in that population, and a statistical method to assess the association between the phenotype and genotype. Over recent decades, much focus has been directed toward "classical" QTL mapping techniques in the mouse, which use phenotypic and genotypic diversity generated using F2 intercrosses or backcrosses and an interval mapping method introduced by Lander and Botstein . This apRecombinant inbred (RI) panels of mice  in whichin silico mapping" . Si. Si17]. loci see . For theAlthough the SMM strategy can be successful at identifying previously known QTL, the biallelic structure of inbred strains at a single SNP locus allows only two genetic groups to be modeled. The assumption of two parental haplotypes is well-suited to the context of QTL mapping based on F2 or RI populations. However, when using the panel of inbred strains of mice, inspection of allele patterns across multiple loci suggests that the genetic structure in many cases is more complex Figure  8-10]. -10. 8-10These observations led us to define an inferred haplotype group as a set of strains with an identical genotype pattern over a local window of SNPs. A genome-wide analysis of inferred haplotype groupings shows that the number of inferred haplotype groups can frequently exceed even four  but rarely exceeds eight (encoded by a three-SNP window) Figure . TherefoUsing these rules, strains are grouped according to inferred haplotype at each three-SNP window. To reduce the occurrence of spurious associations, we require that two inferred haplotype groups of at least five strains each exist at a given locus to be considered. Groups consisting of less than three strains are not considered in the analysis, and strains with missing data are removed from the analysis at this locus. Based on these groupings of inferred haplotype, the F-statistic from analysis of variance (ANOVA) tests the significance of the genotype/phenotype association at a given locus.Given this model of genotypic structure, we first chose to estimate p-values using an inferred haplotype parametric (IH-P) approach based on a parametric model of ANOVA Figure . Here ouFigure The results of association mapping using the IH-KW method are shown in Figure Since rank-based methods discard the quantitative information present in the data, we next examined the use of a nonparametric inferred haplotype bootstrap (IH-B) method to calculate association scores Figure . In contIt is important to note that association scores only indicate candidate genomic regions that may contain a gene that influences a given phenotype. The causal relationship between a specific gene and a phenotype can only be confirmed using experimental techniques . A method of assessing power, the false discovery rate, and/or the false positive rate of our method would therefore be of practical value for evaluating this haplotype association mapping algorithm.Accurate estimates of power for our haplotype association method have not yet been determined since the distributions that arise from our methodology in many cases do not satisfy parametric assumptions of existing methodology in the literature. Methods proposed to estimate the FDR ,19 have While the assessment of false positive rate is less desirable than estimates of power or FDR, it is the only assessment for which a mathematically justifiable approach exists for its computation. The method we utilize applies to multiple testing situations yet makes no assumptions about the independence of the multiple hypothesis tests.k = 1). Given a set of hypothesis tests, the FWER threshold is defined by the probability of observing even one false positive (Type 1 error) occurring among any of the hypothesis tests . The bootstrap process outlined in Figure We implemented an algorithm to compute a family-wise error rate (FWER) of our association scores ,21 Figu, k = 1. It has been understood for some time that the FWER is an extremely strict method of defining the critical value for the rejection region . Using ak+1 Type I errors occurring among any of the hypothesis tests }Since these phenotypes are even less well-characterized than the examples we used in the method development (HDLC and taste preference), detailed analyses of specificity and sensitivity are not possible. Nevertheless, some general trends are apparent. First, more than ten significant loci  by any method were observed in less than half (15 out of 34) of the phenotypes examined. Several factors could explain the lack of associations in the majority of phenotypes. There could be a gene or genes that truly affect the phenotype, but the genotyping data in this region has missing data or is not dense enough to accurately represent the haplotype block structure. Alternatively, the marginal affect of any one gene on the overall phenotype could be too small to detect using these methods. It is also observed that regions of greater haplotype diversity are not interrogated well by these methods due to the loss of power resulting from decreased populations in each haplotype group.Second, in cases where more than ten loci are observed, the IH-KW method is usually the method which reports the largest number of associations. These associations are likely due to the population structure effects described above. Although the modified F-statistic used in the IH-P and IH-B methods accounts for haplotype groups that are dominated by closely related strains, the Kruskal-Wallis statistic is not easily modified in a similar way. Therefore, it is likely that the high number of associations is a result of association to the background genetic structure among the inbred lines. Two other observations corroborate this hypothesis. The use of an unweighted F-statistic also increases the number of observed associations, and the discrepancy is most notable in phenotypes  in which the group of strains with a highest degree of genetic similarity  contains clustered and extreme values (data not shown). In the most extreme example, NEUT, all methods report unusually long stretches of associating loci, indicating that even the modified F-statistic is not sufficient to counteract the strong correlation between phenotype and population structure.QTL mapping in rodents has been an important strategy for narrowing the expansive genome to relatively small regions of the genome containing genes relevant to a phenotype of interest. To date, the majority of QTL have been identified using populations based on F2 crosses. However, these methods are time-consuming and expensive. Furthermore, only a small percentage of the QTL identified using F2 crosses have been mapped down to the causative gene or polymorphism, at least in part due to the comparatively large size of the QTL regions.in silico. Second, large-scale genotyping efforts can be generated and combined in a phenotype-independent manner, making this approach amenable to collaborative efforts that will benefit the entire mouse genetics community. Furthermore, given the existing data sets that we and others have produced, our haplotype association mapping method allows association studies to be quickly performed over a number of available phenotypes .Here, we presented a comparative analysis of methods that utilize the genetic and phenotypic diversity present in common laboratory inbred mouse strains. The potential advantages of this type of approach are two-fold. First, phenotype-specific mouse crosses are not required to generate the required genetic and phenotypic diversity for initial QTL identification. Phenotype data still need to be measured on the panel of inbred mouse lines, but assuming an appropriate range of phenotype values exists, the rest of the association analysis can be performed QTL mapping has also been performed using RI lines, which also have the benefit of combining genotype data in community efforts. However, because commonly available RI lines are derived from only two parents, regions in which the parental strains are identical-by-descent (IBD) cannot be probed for QTL. For example, when comparing C57/BL6J and DBA (parents in the BXD RI panel), only 6292 loci have a different inferred haplotype. In contrast, the full panel of laboratory inbred mouse strains interrogates 11182 loci, even after filtering out loci with trivially small haplotype group sizes. In addition, QTL mapping by RI lines is also currently constrained by the limited availability of specific crosses.Although the set of inbred mouse strains used in our analysis contains greater genotypic and phenotypic diversity compared to the currently available RI lines, the proposal by the Complex Trait Consortium to create additional 1000 RI strains could serve as an even more powerful resource for genome-wide association algorithms . Since tHere, we have explored variants of the association mapping algorithm we originally reported  using diDespite the potential advantages of haplotype association mapping, the limitations in the experimental design relative to traditional cross-based QTL mapping must be noted. As noted above, there is significant population structure in these inbred mice that is not present in either F2 or RI populations. This structure complicates the analysis, and in some cases prevents this strategy from being meaningfully applied to certain phenotypes. The association metric of our haplotype associationmethod also uses a relatively simple ANOVA model (in comparison to more complex maximum likelihood estimation). Traditional linkage analyses base their estimates on regression models that incorporate individual animals, whereas our ANOVA methodology utilizes strain means. Further, sizes of haplotype groups are small compared to the much larger number of individuals utilized in typical linkage studies. All of these factors can lead to a loss of power.et al. [et al. [It is also important to note the strong dependence this haplotype association mapping method has on the inferred haplotype block structure in the mouse genome. While the existence of haplotype block structure is generally accepted, there is ongoing debate regarding the size of these blocks and the ability of haplotype association mapping methods to detect associations. More recent results of Frazer et al.  and Yalc [et al.  indicateRegardless of the relative strengths and weaknesses between haplotype association and traditional QTL mapping, these methods are intermediate steps in pursuit of the final goal – identification of a gene which directly affects the phenotype of interest. In this study, we have used the HDLC levels as the primary phenotype to assess the performance of our algorithms. This phenotype was chosen because it has been extensively studied and many QTL have been previously identified. However, the list of QTL that influence HDLC levels are certainly not exhaustive, and in most cases the specific genes in the QTL regions have not been identified. While the ability of haplotype association methodologies to replicate loci identified in traditional QTL methods is encouraging, this comparison is not an ideal method to assess its specificity and sensitivity. Ultimately, a comprehensive assessment between these approaches may come only after the genetic basis for multiple complex traits has been exhaustively studied.The use of inbred strains of mice for QTL mapping has many advantages over traditional methods. However, there are also limitations in comparison to the traditional linkage analysis from F2 and RI lines, and application of these methods requires careful consideration of algorithmic choices based on both theoretical and practical factors. Here, we have demonstrated that the optimal choice of test-statistic depends on the structure of both the phenotypic and genotypic data, that the use of multi-SNP windows to infer local haplotype structure is essential when using this diverse population of inbred mouse strains, and that the gFWER approach is an effective way to control for multiple testing error while still preserving sensitivity.All authors participated in the design of the algorithms and interpretation of results. In addition, PMC performed all statistical analyses, and PMC and AS drafted the manuscript.Results of single-marker mapping (SMM) method for sweet taste preference. The upper bar chart shows the computed HDLC phenotype association profile using the parametric SMM method. The lower bar chart shows the location of Tas1R3, a gene which has been previously shown to influence sweet taste preference [eference . The x-aClick here for fileResults of inferred-haplotype parametric (IH-P) method for sweet taste preference.Click here for fileResults of inferred-haplotype Kruskal-Wallis (IH-KW) method for sweet taste preference.Click here for file


The development of an instrument accurately assessing service quality in the GP Exercise Referral Scheme (ERS) industry could potentially inform scheme organisers of the factors that affect adherence rates leading to the implementation of strategic interventions aimed at reducing client drop-out.A modified version of the SERVQUAL instrument was designed for use in the ERS setting and subsequently piloted amongst 27 ERS clients.Test re-test correlations were calculated via Pearson's 'r' or Spearman's 'rho', depending on whether the variables were Normally Distributed, to show a significant  relationship between all items within the questionnaire. In addition, satisfactory internal consistency was demonstrated via Cronbach's 'α'. Furthermore, clients responded favourably towards the usability, wording and applicability of the instrument's items.REFERQUAL is considered to represent promise as a suitable tool for future evaluation of service quality within the ERS community. Future research should further assess the validity and reliability of this instrument through the use of a confirmatory factor analysis to scrutinise the proposed dimensional structure. Exercise Referral Schemes (ERSs) in the UK involve a recommendation from a clinician to a patient concerning the up-take of physical activity, based upon certain pre-determined criteria. The patient then attends a local exercise centre, where an advanced exercise instructor will create an appropriate individualised programme, usually designed to last around 12–15 weeks. The publication of 'Exercise Referral Systems: A National Quality Assurance Framework' . Client • Tangibles: Physical facilities, equipment, and appearance of personnel.• Reliability: Ability to perform the promised service dependably and accurately.• Responsiveness: Willingness to help customers and provide prompt service.• Assurance: Knowledge and courtesy of employees and their ability to inspire trust and confidence.• Empathy: Caring, individualised attention the firm provides its customers.The SERVQUAL instrument comprises twenty two questions tailored to assess customers' expectation of service quality, followed by a further twenty two items designed to identify the clients' perception of the same issues. Each item is scored on a seven point Likert scale, thus allowing the equation Q = P - E to be calculated .et al. [Since PZB's  publicatet al.  go as fai.e. without modification) to any service is widely criticised in the literature [Applying SERVQUAL in its pure form , offering the researcher greater insight into whether the service quality of the operational aspects of the scheme or the overall management of the referral site may have most critically affected adherence.An adaptation of the Blair et al.  7-day PhConsidering the lengthy and relatively complex nature of SERVQUAL, the 7PAR was simplified from five to three levels of physical activity , although duration of exercise was retained. Definitions and examples of light, moderate and vigorous physical activity were given.Many client-related factors established as being significant determinants to adherence in the literature, formed the foundation from which tailoring of the SERVQUAL instrument was undertaken. Part of the tailoring of the SERVQUAL instrument involved the creation of two new dimensions as a number of topics raised in the literature did not conceptually 'fit' into the existing dimensional framework. The first of these relates to the relationship between the client and the GP – an association distinct to the ERS industry from any previously examined. The second relates to personal perceptions of exercise. The existing 22 items of SERVQUAL were supplemented with the questions see .Modification of SERVQUAL to eliminate negatively-worded questions included in the original instrument has received unanimous support, this was also taken into account when tailoring the items to the ERS Setting .The pre-pilot REFERQUAL appended with a feedback sheet was distributed to six attendees at five ERSs and also to the corresponding scheme organisers. The five ERSs had been selected to participate in the main post-pilot study. Feedback was received with respect to the instrument's ease of use, wording and any other topic the respondents felt relevant. Some questions were subsequently removed the instrument whilst others were modified following feedback to enhance applicability and clarity or to eliminate duplicity.Subsequently, one exercise class comprising 30 clients was invited to participate in the pilot study. The Exercise Professional leading the group distributed REFERQUAL by hand and responses were completed whilst the exercisers were at the centre. The Exercise Professional subsequently repeated the operation two weeks later with the same group. Twenty-seven of the 30 in the group completed both questionnaires, a response rate of 90%, the other three participants were absent for one of the two sessions. Again, respondents were also invited to comment on the instruments ease of use, wording and any general concerns relating to REFERQUAL.In order to determine test re-test reliability, correlation will be calculated via Pearson's 'r' or Spearman's 'rho' depending on the distribution of the individual items. One sample Kolmogorov-Smirnov (K-S) tests will be used to determine whether items are drawn from normally distributed data. K-S testing is a suitable measure of distribution regardless of sample size , is consK-S tests were conducted on each of the 35 perception/expectation scores elicited from each completion (p < 0.05). In cases where both item scores were drawn from normally distributed data, correlation was calculated via Pearson's "r" . The two dimensions scoring slightly lower than the others (responsiveness and tangibles) are not considered to give cause for concern. Responsiveness is widely acknowledged to be a fundamental contributor to the understanding and assessment of service quality ,37 and RHowever, the applicability of tangibles as a dimension within service quality assessment has received criticism, relating to client's perception of the concept as a proxy for evaluating service outcomes . HoweverCalculations within this investigation have been made via the perception/expectation score generated by the SERVQUAL format. Future studies should analyse both perception and expectation items separately to confirm the validity and reliability of this model particularly bearing in mind the fundamental advantage of the perception/expectation 'gap' model over the performance-only instruments – that the perceptions of those clients not visiting the referral sites at all could be gathered. This investigation solely sought the views of active exercises at various stages of completion of referral. Future studies should seek to investigation across the entire A-E spectrum outlined above.Preliminary results indicate REFERQUAL to represent a promising model of service quality assessment within the ERS setting. Correlational findings are extremely supportive and are underlined by satisfactory reliability scores. However, future studies incorporating the views of the entire adherence spectrum featuring far greater sample sizes and subsequent factor analysis on the dimensional structure of REFERQUAL will offer far greater insight into the appropriability of this new model.The author(s) declare that they have no competing interests.DC: Responsible for the overall study design and the acquisition and analysis of the data. DC was also responsible for the drafting of the document.IA: Made substantial contributions to the conceptual design of the study and responsible for considerable drafting input.AI: Made substantial contributions to the conceptual design of the study and responsible for considerable drafting input. Also responsible for statistical guidance and advice.PB: Made substantial contributions to the conceptual design of the study and responsible for considerable drafting input.The pre-publication history for this paper can be accessed here:Figure 1, The SERVQUAL Instrument.Click here for fileFigure 2, Supplementary Questions Added to SERVQUAL.Click here for file


A large number of fatigue scales exist and there is no consensus on which fatigue measuring scales that are most appropriate for use in assessment of fatigue in different diseases. We aimed to describe the use of fatigue scales in studies of disease-related fatigue during the last three decades. We searched databases from 1975 to 2004 for original studies reporting on disease-related fatigue and extracted information on method used to assess fatigue, diseases under study and year of publication. A total of 2285 papers reported measures of fatigue in chronic non-acute diseases of which 80% were published during the last decade. We identified 252 different ways to measure fatigue, of which 150 were use only once. Multi-symptom scales (n = 156) were used in 670 studies, while 71 scales specifically designed to measure fatigue were applied in 416 studies. The majority of these studies used scales with a multidimensional approach to fatigue, and most studies used scales that were disease-specific or only applied to few different diseases. Research in disease-related fatigue has increased exponentially during the last three decades, even if we adjust for the general increase in publishing activity. The number of scales has also increased and the majority of scales were developed for specific diseases. There is need for measure instruments with different sizes and dimensionality, and due to ceiling and floor effects, the same scale may not be useful for patients with different severity of fatigue. However, since fatigue is an unspecific symptom there should not be need for adopting disease specific fatigue scales for each individual disease. There may be differences in characteristics of fatigue between diseases and generic measurement instruments may facilitate documentation of such differences, which may be of clinical importance. Fatigame sick . Such fiame sick . Fatiguee scales .Fatigue scales have recently been reviewed elsewhere ,7. DittnWe searched MEDLINE and PsycINFO for studies reporting on disease-related fatigue in the period 1975 to 2004. The search was last updated February 2004. MEDLINE was searched using the MeSH terms 'fatigue' or 'asthenia', supplemented with a free text search for the terms 'fatigue', 'asthenia' or 'tiredness' occurring together with the MeSH terms 'questionnaires', 'health surveys', 'epidemiologic studies', or 'quality of life'. The PsycINFO database was searched for articles with the descriptor 'fatigue' occurring together with classification codes indicating somatic, functional or psychiatric disease . Several check-ups in reference lists suggested that inclusion of other databases would only result in few additional peer-reviewed studies of fatigue in populations of patients.References without abstract and articles written in non-English language were excluded. All references and abstracts were imported to a database and the following information was extracted from the abstract: method used to assess fatigue, disease(s) under study and year of publication. If it was not possible to extract this information from the abstract, the full-text paper was reviewed. Ad hoc constructed questions and review of medical records were grouped together (ad hoc methods), so was one-item questions used to assess acute treatment side effects. All original studies in adult populations of patients with specified non-acute disease were included in the analyses.quality of life scales and other multi-symptom scales were introduced, while scales specifically designed to measure fatigue have mainly been in use the last decade  declare that they have no competing interests.NHH designed the study, carried out the literature review and data analyses and drafted the manuscript. JHA and PB commented and discussed the analyses and the manuscript. All authors read and approved the final manuscript.


Our objective is to test the ability of an Ultraseek® search engine to rank MEDLINE® records of the included studies of Cochrane reviews within the top half of all the records retrieved by the Boolean MEDLINE search used by the reviewers.Most electronic search efforts directed at identifying primary studies for inclusion in systematic reviews rely on the optimal Boolean search features of search interfaces such as DIALOGCollections were created using the MEDLINE bibliographic records of included and excluded studies listed in the review and all records retrieved by the MEDLINE search. Records were converted to individual HTML files. Collections of records were indexed and searched through a statistical search engine, Ultraseek, using review-specific search terms. Our data sources, systematic reviews published in the Cochrane library, were included if they reported using at least one phase of the Cochrane Highly Sensitive Search Strategy (HSSS), provided citations for both included and excluded studies and conducted a meta-analysis using a binary outcome measure. Reviews were selected if they yielded between 1000–6000 records when the MEDLINE search strategy was replicated.Nine Cochrane reviews were included. Included studies within the Cochrane reviews were found within the first 500 retrieved studies more often than would be expected by chance. Across all reviews, recall of included studies into the top 500 was 0.70. There was no statistically significant difference in ranking when comparing included studies with just the subset of excluded studies listed as excluded in the published review.The relevance ranking provided by the search engine was better than expected by chance and shows promise for the preliminary evaluation of large results from Boolean searches. A statistical search engine does not appear to be able to make fine discriminations concerning the relevance of bibliographic records that have been pre-screened by systematic reviewers. Systematic reviews in healthcare are designed to summarize and synthesize the existing totality of evidence about a clinical question using rigorous methods that help to safeguard the review from bias. Ideally, the evidence-gathering stage requires preparing as broad a search 'statement' as possible, and then adjusting the electronic search parameters to capture just the right kind of evidence to be useful without overburdening reviewers. More than three quarters of the studies included in systematic reviews are identified through electronic bibliographic databases that are searched using Boolean logic -5. BooleSearch engines can rank search results by relevance based on where in the document the keywords appear, and how often. Using such a search engine to assign relevance to the large result set of a comprehensive Boolean search could reduce the amount of material requiring expert attention, if the documents that meet the strict eligibility criteria of the review are highly ranked.Typically, the manual review of the results of Boolean electronic searches is often done in a two-stage process. In the first stage, reviewers may work from the bibliographic record, or document title, and screen out those that are obviously irrelevant to the review. In the second stage, reviewers usually obtain the full articles associated with remaining records and then decide eligibility based on the complete report, rather than on the more limited information available at the first stage of screening.We evaluated a two-step approach consisting of a comprehensive Boolean search followed by automated relevance ranking for eligible systematic reviews from the Cochrane Library (Cochrane reviews henceforth) to explore the feasibility of such an approach.rd Quarter 2002 was searched through the Ovid interface using the search string (hsss or highly sensitive search).tw. to identify potential studies. Two reviewers assessed each systematic review against the eligibility criteria and resolved any conflicts through consultation. SRS™ was used for all screening and data extraction. SRS is a web-based platform for conducting systematic reviews [We sought Cochrane reviews that used at least one phase of the HSSS  to ident reviews .The size of the MEDLINE retrieval was determined by replicating and running the MEDLINE search strategy. Cochrane reviews with a MEDLINE retrieval of 1000–6000 records were selected for testing the performance of the Ultraseek search engine ranking.One librarian extracted descriptive data about the eligible reviews. The following elements were recorded: the number of included studies and the number of excluded studies cited in the review; the number of included studies indexed in MEDLINE; the number of excluded studies indexed in MEDLINE; date of the MEDLINE search reported in the body of the review; level of detail in which the search was reported; phases of the HSSS used; searching techniques employed ; and restrictions such as date, language of publication, age groups or methodological filters. Electronic databases searched as well as other sources used (such as checking reference lists or contacting authors or manufacturers) were also recorded.A known-item search was undertaken in MEDLINE for each included and excluded study listed in the review. A single librarian (MS) completed the searching using the Ovid interface for MEDLINE 1966-April 2003. The indexing status of each study was recorded as indexed or not indexed, and for each review, the set of included studies was aggregated using OR statements, as was a set of excluded studies. Each set was downloaded for subsequent analysis.The Ovid bibliographic records for all studies retrieved from MEDLINE by the replicated search were also downloaded. When the review reported the size of the MEDLINE retrieval, it was compared to ours to validate the replication. Where our search result was smaller than that reported in the review, it was excluded as irreproducible.Produced by Verity, Ultraseek was originally a successful web search engine and is now focused on helping businesses manage their digital information . The UltThe Ultraseek search engine indexes "collections". The bibliographic records associated with each systematic review were treated as a collection. Bibliographic records were downloaded from MEDLINE into Reference Manager databases, and tagged according to their inclusion status in the review. A Reference Manager output format was created to write each record with HTML tags. Three sets of fields were written as meta-data – MeSH headings, title and abstract . A Perl script was used to separate the HTML tagged bibliographic records into individual files. File names encoded the ID number of the review, whether the record was included or excluded from the review, and the reference ID number within that review, in the form http://10included3.html. Thus the collection consisted of the bibliographic records re-written as HTML files tagged with meta-data. The search engine was installed on a laptop computer where the collections resided. The search engine indexed all records in the collection. When a search was run against the collection, the number of items with relevance greater than zero was returned, along with list of up to the first 500 relevant items, sorted by relevance.The Ultraseek search engine was configured to provide weights to the meta-data fields – index terms, title and description (abstract). When the weights given to the meta-data fields were varied in preliminary testing the relevance scores changed, but not the order of items, which was the variable of interest. Thus, the search engine was configured with all elements equally weighted, and the collections were indexed.For each eligible review, one member of the research team (MS) identified subject terms to be entered into the Ultraseek search. In exploratory work, it became apparent that the number of tied relevance scores depended largely on the number of terms entered. Thus we decided to standardize our Ultraseek searches at 7 terms, the minimum number that seemed to reduce ties to a workable number.We also established that the order in which terms were entered influenced the final relevance score. Terms were entered on the basis of perceived importance  the rankings of included studies within a collection comprising the entire MEDLINE retrieval and ii) the ranking of included studies where only the studies listed as included or excluded in the Cochrane report comprised the collection. As we were concerned that the search engine might be optimized to place highly relevant items in the top few items with less exact ranking further back in the pack, we alsoWhen testing the initial MEDLINE retrieval, recall was determined by considering the proportion of included studies ranking within the top 500. We compared the proportion falling within the top 500, based on their relevance rank, with the proportion expected if ranking was random. When testing listed included and excluded studies, the rankings were analyzed with a Wilcoxon rank sum test using SAS for exact permutations, in order to best handle the small data sets and frequent ties .One hundred and sixty nine Cochrane reviews were retrieved and screened for eligibility. Sixty-four of them were excluded either because they do not use any phase of the HSSS or they did not report the citations of included and excluded trials. The remaining 105 reviews met our inclusion criteria .For three reviews where the Ultraseek ranking performed poorly, we attempted another search, using a different set of terms. Terms and performance on each of the two attempts are reported in Table There was no statistically significant difference between the rankings of studies listed as included versus studies listed as excluded . Further, no obvious pattern between the included and excluded studies could be discerned through visual inspection  a relevance-ranking search engine will have difficulty distinguishing relevant from irrelevant items on the basis of the information contained in the bibliographic record.The approach tested here is interesting as a proof of concept. Truncating the search result at 500 records would have resulted in great efficiencies, reducing the number of records to be reviewed from 26499 to 4500 across the nine reviews, but at the expense of losing 30% of relevant studies – few reviewers would likely accept such a trade-off.Incorporating additional information into the rankings might facilitate any future exploration of using search engines to reduce the time taken to conduct a systematic review. There are at least four potential sources of additional information. First, giving increased weight to information in the title and index terms is likely to improve performance; we were not able to do this through meta-data weightings. A second source of information to improve accuracy would be the full document. Our results were based on the information contained in a document surrogate – the bibliographic record. A full text approach will become practical as a larger proportion of articles considered for inclusion in systematic reviews become available electronically.A third source of additional information would be the prior decisions of the reviewers. Novel techniques developed by de Bruijn and Martin  have beevs. excluded lists could not be distinguished points to a role for ranking at the broad screening stage of the review. We did not see a strong enough concentration of relevant records in the top 10 to suggest that some records were so clearly relevant that they should be promoted into the review without manual examination. A useful step would have been to establish a lower cut-off point, below which we could be confident that no relevant studies would fall. This would have permitted us to exclude the lowest ranking (including non-ranked) records without subjecting them to any manual review, thereby gaining efficiency. The search engine selected for the experiment did not enable us to examine any but the first 500 hits.Rankings could be used in several ways. Our finding that included studies receive higher than chance rankings but that the included ® records into HTML format. Ranking holds promise and future research is warranted to explore methods that incorporate more information into the relevance ranking process. A system reliably placing the relevant records within a smaller ranked set derived from the Boolean retrieval can speed the formation of the evidence base for systematic reviews in healthcare.Relevance ranking of Boolean search results is technically feasible using a commercial search engine under academic license to index and search collections built through a simple procedure to render Reference ManagerSample bibliographic record showing HTML markup and meta data tags<meta name="author" content="Fabacher, D., Josephson, K., Pietruszka, F., Linderborn, K., Morley, J. E., and Rubenstein, L. Z."><meta name="title" content="An in-home preventive assessment program for independent older adults: a randomized controlled trial. -see comments.-"><meta name="subjects" content="Activities of Daily Living;Aged;Comparative Study;Female;Follow-Up Studies;Geriatric Assessment;Health Status;Home Care Services;Human;Male;Non-U.S.Gov't;Patient Compliance;Preventive Health Services;og -Organization & Administration-;Support;United States;Veterans;Voluntary Workers"><meta name="Abstract" content="OBJECTIVE: To evaluate the effectiveness of in-home geriatric assessments as a means of providing preventive health care and improving health and functional status of ... truncated for illustration ... aspects of health and function"><title>An in-home preventive assessment program for independent older adults: a randomized controlled trial. -see comments.-</title>
Ref ID = 3

Fabacher, D., Josephson, K., Pietruszka, F., Linderborn, K., Morley, J. E., and Rubenstein, L. Z. An in-home preventive assessment program for independent older adults: a randomized controlled trial. -see comments.-.Journal of the American Geriatrics Society 1994;42(6):630–638.

MeSH Subject Headings:
Activities of Daily Living;Aged;Comparative Study;Female;Follow-Up Studies;Geriatric Assessment;Health Status;Home Care Services;Human;Male;Non-U.S.Gov't;Patient Compliance;Preventive Health Services;og -Organization & Administration-;Support;United States;Veterans;Voluntary Workers
Abstract:
OBJECTIVE: To evaluate the effectiveness of in-home geriatric assessments as a means of providing preventive health care and improving health and functional status of ... truncated for illustraton ... aspects of health and function
The author(s) declare that they have no competing interests.MS conceptualized the project, screened records for eligibility, undertook data collection and analysis, created the Reference Manager output format, selected search terms, prepared the first draft of the manuscript, and participated in all revisions. NJB obtained funding for the project, designed the statistical analysis and created the Perl scripts, and participated in the drafting and revision of the manuscript. DM obtained funding for the project, advised on the design and conduct of the experiments and participated in all revisions of the manuscript. TJC acted as project leader for the grant, advised on the design and conduct of the experiments and participated in all revisions of the manuscript. RWP obtained funding for the project, advised on the statistical analysis, the design and conduct of the experiments and participated in all revisions of the manuscript. AM screened records for eligibility, undertook data collection, and participated in all revisions of the manuscript. TK obtained funding for the project, the design and conduct of the experiments and participated in all revisions of the manuscript. LZ configured the search engine, replicated the searches, undertook known item searching, built and indexed the collections, created the datasets, and participated in all revisions of the manuscript.All authors reviewed and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Mus musculus, as evidenced both by recent and extensive gene duplication and by high ratios of nonsynonymous to synonymous nucleotide substitution rates. This makes ABP an appropriate model system with which to investigate how recent adaptive evolution of paralogous genes results in functional innovation .The genes for salivary androgen-binding protein (ABP) subunits have been evolving rapidly in ancestors of the house mouse Abp paralogues in the mouse genome as possible. We observed expression of six Abpa paralogues and five Abpbg paralogues in ten glands and other organs located predominantly in the head and neck . These Abp paralogues differed dramatically in their specific expression in these different glands and in their sexual dimorphism of expression. We also studied the appearance of expression in both late-stage embryos and postnatal animals prior to puberty and found significantly different timing of the onset of expression among the various paralogues.It was our goal to find evidence for the expression of as many of the Abpa/Abpbg expression in different glands; and 2) sexual dimorphism in the expression of the paralogues in a subset of those glands. These expression patterns differ dramatically among various glands that are located almost exclusively in the head and neck, where the sensory organs are located. Since mice are nocturnal, it is expected that they will make extensive use of olfactory as opposed to visual cues. The glands expressing Abp paralogues produce secretions  or detect odors (MOE and VNO) and thus it appears highly likely that ABP proteins play a role in olfactory communication.The multiple changes in the spatial expression profile of these genes resulting in various combinations of expression in glands and other organs in the head and face of the mouse strongly suggest that neofunctionalization of these genes, driven by adaptive evolution, has occurred following duplication. The extensive diversification in expression of this family of proteins provides two lines of evidence for a pheromonal role for ABP: 1) different patterns of  Genome sequences of primates and rodents -3 now alThe process of gene duplication provides material for functional diversification. A newly-duplicated gene may subsequently acquire innovative function  or may retain some of its progenitor gene's functional repertoire , or a combination of both -11. SuchDrosophila development. We wish to ask about diversification during other evolutionary process, to wit rapidly diverging duplicated genes involved in the four functions listed above. Because expression patterns appear to diverge substantially over long time periods, such as since the primate-rodent (Euarchontoglires) common ancestor Abp paralAbpa/Abpbg expression begins in the head of the embryo as early as 13 days of gestation, comprised of a relatively simple pattern of a11/bg11 and bg10. By the first day of life, this has changed little, but by six days many other paralogues are expressed in the submaxillary and lacrimal glands. Expression proceeds to 15 days of life without yet revealing the sexually dimorphic patterns seen in several glands in adult mice. The transition to sexually dimorphic expression following puberty appears incompatible with roles for these in host immune defense.The developmental sequence of expression of these genes provides clues that help further narrow the choices of functional role for ABP. It is fascinating that the MOE and VNO are the last tissues to achieve an adult expression pattern. Unlike the lacrimal gland and the salivary glands, these are not secretory tissues. It may be that early expression in the pups' secretory tissues provides signals by which the female parent recognizes her offspring. The pups would not need MOE and VNO expression themselves at this early stage. This distinction hints at a role for ABP in chemosensation, perhaps by participating in detection and recognition of ABP subtypes. Paralogues expressed in the MOE and VNO could be involved in phenotype matching and/or in a system whereby a bound ligand is transferred between paralogues to augment the signal of a proteinaceous pheromone.Abp evolution. We previously reported finding a single, pseudogenized Abp gene pair in both the human and the chimpanzee genomes, while the genomes of the mouse and rat had approximately 14 and 3 pairs of Abp genes, respectively, most of which had full-length ORFs [Abp gene pair in each of the dog and cat genomes would be consistent with a pattern of increasing importance of Abp genes in conspecific communication, proceeding from potentially no importance in primates to low importance in carnivores to relatively high importance in murid rodents, with the mouse utilizing the most number and diversity of Abp gene pairs.Recently, Grus et al.  studied gth ORFs . A singlAbpa/Abpbg paralogues we predicted. In this report, we have shown that Abp genes have evolved multiple, often distinct, expression patterns. Once these patterns are mapped to these genes' proposed phylogenetic tree a surprising lack of congruency is observed  sexual dimorphism in the expression of the paralogues in a subset of those glands. It is clear from our observations that expression patterns of many of the Abp paralogues differ dramatically among various glands that are located almost exclusively in the head and neck, where the sensory organs are located. Since mice are nocturnal, it is expected that they will make extensive use of olfactory as opposed to visual cues. The glands expressing Abp paralogues produce secretions  or detect odors (MOE and VNO) and thus it appears highly likely that ABP proteins play a role in olfactory communication. This supports the earlier suggestion, based on behavioral testing, that salivary ABP  mediates mate preference [The extensive diversification in expression of this family of proteins provides two lines of evidence for a pheromonal role for ABP: 1) different patterns of eference ,30. In teference , femaleseference .Abp paralogues act as components of a complex pheromone system, then the secretions of the lacrimal gland and salivary glands provide a constellation of olfactory information, such as an animal's age and sex. Indeed it has been proposed previously that polymorphism in salivary ABP communicates the species/subspecies of the animal [Abp paralogous genes in adaptive evolution is consistent with a pheromonal role for their protein products.If the variety of proteins produced by expression of the mouse e animal -32,49. TAbpa and Abpb . Thus, regulation of the expressions of Abpa and Abpbg members of pairs must be more complex than simple coordinate up- or down-regulation of pairs. The lack of correlation between the evolutionary relationships of the Abpa/Abpbg paralogues [Abpa/Abpbg paralogues.Laukaitis et al.  proposed bg11 in  and in t bg11 in  suggesteralogues  and theiAbpa/Abpbg genes in mice. This is perhaps an even more striking example because the single orthologous pair of Abpa/Abpbg genes in primates both degenerated into pseudogenes while expression in rodents has expanded into at least 9 tissues from the 2 tissues reported for the cat [Huminiecki and Wolfe  studied  the cat .Abpa/Abpbg gene pair was pseudogenized, suggesting that its function has become dispensable. Many genes involved in olfactory and pheromonal cues have become pseudogenes in primates [The multiple changes in the spatial expression profile of these genes resulting in various combinations of expression in glands and other organs in the head and face of the mouse strongly suggest that neofunctionalization of these genes, driven by adaptive evolution, has occurred following duplication. Primates represent an interesting contrast insofar as their single primates . EvolutiAbpa paralogues and 13 Abpbg paralogues in mouse strain C57BL/6. Five of the Abpa paralogues and five of the Abpbg paralogues were predicted to be pseudogenes because of frameshifts and/or termination codons occurring early in their sequences. Primer sets were designed to specific forward and reverse sequences for the remaining nine Abpa and eight Abpbg paralogues. Primers were designed to avoid cross-hybridization between paralogues' sequences. Primer sets were purchased from Sigma-Genosys . DNA from strain C57BL/6 was obtained from Jackson Laboratory for use in testing the primer sets.The C3H/HeJ inbred strain of mice was purchased from Jackson Laboratory . Mice at age 75–80 days were sacrificed by cervical dislocation; tissues and organs were removed and frozen immediately in liquid nitrogen and thereafter stored at -80°. Emes et al.  publisheRNA was purified from mouse tissues with a Sigma total RNA purification kit . Absorbances at 260 and 280 nm were determined and used to calculate yield, purity and concentration of RNA from tissue extractions. In some instances, the RNA was treated with RNase-free DNase . Oligo-dT was used to prime first-strand synthesis from 1 μg of total RNA per 20 μl RT reaction essentially as described in  using SiClean-up of PCR templates was performed using QiaQuick PCR clean-up spin columns  according to the manufacturers protocol (Qiagen Incorporated) and automated sequencing was performed by ACGT, Inc. .This work arose from a comparative genomic analysis in which RDE, RCK, CML and CPP agreed to divide the bioinformatics analyses (published in 2004) and the expression analyses (this paper) between the two laboratories, Ponting's at Oxford University and Karn's at Butler University. SRD directed and participated in the harvesting of the many mouse tissues required for the study and he contributed to the design of the expression detection experiments. CML and RCK carried out the PCR expression analyses. RDE and CPP created the annotated gene trees. All authors made substantive intellectual contributions to this work and all authors read and approved the final manuscript.


Gene regulation and metabolic reactions are two primary activities of life. Although many works have been dedicated to study each system, the coupling between them is less well understood. To bridge this gap, we propose a joint model of gene regulation and metabolic reactions.We integrate regulatory and metabolic networks by adding links specifying the feedback control from the substrates of metabolic reactions to enzyme gene expressions. We adopt two alternative approaches to build those links: inferring the links between metabolites and transcription factors to fit the data or explicitly encoding the general hypotheses of feedback control as links between metabolites and enzyme expressions. A perturbation data is explained by paths in the joint network if the predicted response along the paths is consistent with the observed response. The consistency requirement for explaining the perturbation data imposes constraints on the attributes in the network such as the functions of links and the activities of paths. We build a probabilistic graphical model over the attributes to specify these constraints, and apply an inference algorithm to identify the attribute values which optimally explain the data. The inferred models allow us to 1) identify the feedback links between metabolites and regulators and their functions, 2) identify the active paths responsible for relaying perturbation effects, 3) computationally test the general hypotheses pertaining to the feedback control of enzyme expressions, 4) evaluate the advantage of an integrated model over separate systems.The modeling results provide insight about the mechanisms of the coupling between the two systems and possible "design rules" pertaining to enzyme gene regulation. The model can be used to investigate the less well-probed systems and generate consistent hypotheses and predictions for further validation. Gene regulation and metabolic reactions are two primary activities of life. Despite vast amount of works have been dedicated to study each system, the coupling between the two systems is relatively less probed. It is therefore necessary to shift focus from individual systems to their integration.The coupling between metabolic reactions and gene regulation is supported by abundant direct and indirect evidence. Some assays indicate substrates of metabolic reactions influence the activities of transcription factors or signal transduction pathways e.g., -4). More. More4])Previous methods of modeling the joint processes of gene regulation and metabolic reactions fall into three general categories. The first approach explicitly establishes the links between specific metabolites and transcription factors and models the influence between metabolites and gene expression. Examples include imposing binary constraints from gene expression data on metabolic fluxes ,11, explIn this work we propose a joint model of gene regulation and metabolic reactions that combine the merits of the first two approaches and compensate their shortcomings. Our model aims for answering the following questions. First, we want to demonstrate whether the coupling between the two systems is essential to explain different types of perturbation data. Second, the identities and functions of the feedback links between metabolites and regulators are often unknown. We want to find those missing links from the perturbation data. Third, we want to identify the mechanisms responsible for perturbation responses. By abstractizing these mechanisms as paths in the joint network of gene regulation and metabolic reactions, this question is translated into finding the "active paths" which explain the perturbation responses. Fourth, in addition to individual  links we are also interested in the "design rules" regarding the architecture and functions of the feedback regulation of metabolic enzymes. We want to test whether certain hypotheses pertaining to feedback regulation can better explain the experimental data. The general concept of our model is illustrated in Figure H2O, CO2, ATP, ADP, cAMP). Fourth, we only consider a special case of the combinatorial effects of multiple transcription factors or enzymes: each factor or enzyme acts independently and the change of each factor or enzyme suffices to alter gene expression or metabolic reactions.We encode these constraints as a probabilistic graphical model – factor graph – over the network attributes, and apply a graphical model learning algorithm to identify the attribute values which optimally fit the data. To build a joint network we use two methods to establish links between metabolites and genes. The first method searches among all  pairs and incrementally augments the joint network with the links which maximize the number of explained perturbation responses. The second method explicitly adds links according to several general hypotheses pertaining to the functions of metabolic pathways and enzymes. The modeling framework is illustrated in Figure We first describe a joint network of gene regulation and metabolic reactions and the datasets used in this work. We then define the terminology pertaining to paths in the joint network and outline the constraint-based probabilistic graphical model over model attributes. We applied two approaches to construct the joint models of metabolic reactions and gene regulation and used them to explain the perturbation responses. The first approach learns the feedback links between metabolites and regulators which best explain the data. We investigated the change of explanatory power with respect to path length and active paths connecting perturbation sources and responses in this joint model. The second approach encodes four general hypotheses pertaining to enzyme gene regulation in the joint model. To further validate the predictive power and the consistency of the model learning algorithm, we did two validation tests. We performed three cross validation tests on the perturbation data to ensure the inferred model could accurately predict the responses in the test data. We also simulated perturbation responses from the models with hypothetical feedback links and verified that the model learning algorithm could retrieve these feedback links from simulated data.Escherichia coli. The regulatory part of the network contains the nodes of regulators (transcription factors), operons and genes. Regulators link to operons they bind and operons link to their member genes. The metabolic part of the network contains the nodes of metabolites, fluxes of reactions, and enzymes. Metabolites at both sides of a reaction connect to the flux of this reaction with bidirectional edges. Enzymes catalyzing a reaction connects to its flux with unidirectional edges. The regulatory and metabolic parts are coupled by two types of directed edges: edges linking genes to enzymes indicating the mapping between enzyme gene identities and functions, edges linking metabolites to regulators indicating the feedback control of metabolites on regulator activities.We constructed the joint network of gene regulation and metabolic reactions of Many metabolic reactions are either irreversible or have a "preferred direction" under the normal growth condition. We therefore annotate the preferred directions of reactions and specify their "input" (substrate) and "output" (product) metabolites. For instance, in reaction glucose + ATP ⇌ glucose-6-phosphate + ADP, glucose is the input and glucose-6-phosphate is the output.E. coli, the substrates and enzymes of metabolic reactions, the operons of many genes, the bindings of transcription factors on some operon promoters, and the functions of these transcription factors were downloaded from the EcoCyc database 29].To evaluate the explanatory power of the model we compare the predicted responses according to the inferred MAP configurations to the observed perturbation data. Given a configuration, for each  tuple we identify the active paths connecting the tuple according to the configuration. We then predict the response along each active path using the procedure described above. A  tuple is explained by a configuration if the predicted responses along all active paths are identical to the actual response. A  tuple is explained by the model if it is explained by all MAP configurations inferred from the model. In contrast, a  tuple is contradicted with the model if the model has a consistent prediction but the consistent prediction is the opposite of the observed response. We thereby evaluate the explanatory power of the model by counting the number of  tuples explained by the model minus the number of  tuples contradicted with the model.The same procedure is also used to evaluate the predictive power of the model in a cross-validation setting. The MAP configurations are inferred from the training data and we check whether the predicted responses along all active paths in each MAP configuration are consistent with the test data. Different methods of splitting the training and test data are described in Results.We apply permutation tests to further justify the significance of the model's explanatory power. The perturbation responses of genes or fluxes in each experiment are randomly permuted. The numbers of permuted  tuples consistent or contradicted with model prediction are counted. The p-value of the model's explanatory power is the fraction of random trials whose (# explained tuples – # contradicted tuples) exceed the empirical value.p > 10-3).We propose two methods to identify and test the coupling between gene regulation and metabolic reactions. The first approach attempts to learn the feedback links between metabolites and transcription factors from the perturbation data. We identify those missing links in terms of their power to explain the perturbation data. Define the explanatory power of a set of  links as the number of explained perturbation responses minus the number of contradicted perturbation responses of the joint network model with those links. Our goal is to find the links which maximize the explanatory power. We apply a simple method of incrementally adding  links which maximize the gain of the explanatory power. Candidate links are the pairs between all metabolites and all regulators. At each iteration we find the link which yields the maximum gain of the explanatory power. When multiple links possess equal explanatory power, we branch the search process by creating multiple joint networks, each with one optimal link added. Edge addition stops when the gain of the explanatory power is statistically insignificant according to the permutation test . For example, to encode hypothesis 4 (glucose repression) we add negative edges from glucose to enzymes in TCA cycle and genes involved in electron transfer. A factor graph model is then built on the augmented joint network to explain the perturbation data.We encoded the programs of model inference, search and validation in C, and ran the programs on a dual-processor Linux PC . The running time for inferring the optimal configurations given the fixed links or general hypotheses varies with the maximum path length. For the maximum path length equal to 6, the running time is less than 10 seconds. The running time for searching the optimal links among all  pairs with maximum path length 6 is about 30 minutes. The running time for leave-one-out, 5-fold or 10-fold cross validation with maximum path length 6 is about 30 minutes. The source codes of these programs and the tabulated forms of the perturbation data are provided in the supplementary files [see Additional files CHY and MV conceived the models. CHY implemented the models, collected and analyzed data with the models, and wrote the paper.Active paths explaining expression changes in glucose reduction, model of inferred links.Click here for fileActive paths explaining expression changes in pykF knock-out, model of inferred links.Click here for fileActive paths explaining expression changes in acetate enhancement, model of inferred links.Click here for fileActive paths explaining flux changes in pykA/pykF double knock-outs, model of inferred links.Click here for fileActive paths explaining flux changes in pgi knock-out, model of inferred links.Click here for fileActive paths explaining flux changes in zwf knock-out, model of inferred links.Click here for fileActive paths explaining expression changes in glucose reduction, model of general hypotheses.Click here for fileActive paths explaining expression changes in pykF knock-out, model of general hypotheses.Click here for fileActive paths explaining expression changes in acetate enhancement, model of general hypotheses.Click here for fileActive paths explaining flux changes in pykA/pykF double knock-outs, model of general hypotheses.Click here for fileActive paths explaining flux changes in pgi knock-out, model of general hypotheses.Click here for fileActive paths explaining flux changes in zwf knock-out, model of general hypotheses.Click here for fileThe number of perturbation responses explained and contradicted by the model of inferred links with varying length, categorized by (1)types of responses (2)magnitudes of responses (3)types of paths explaining the responses.Click here for fileThe number of perturbation responses explained and contradicted by the model of general hypotheses with varying length, categorized by (l)types of responses (2)magnitudes of responses (3)types of paths explaining the responses.Click here for fileThe source codes of the model inference programs.Click here for fileE. coli central carbon network and perturbation experiments.The data of the Click here for file