Despite significant progress in treatment, chronic lymphocytic leukemia (CLL) remains an incurable disease. Advances have been made to understand the molecular pathogenesis underlying CLL progression and treatment resistance. We here review the available evidences concerning the role of the B-cell receptor (BCR) and the tumor microenvironment interactions in CLL pathogenesis. Antigen likely has a key role in the selection of the tumoral clone, the mutational status of immunoglobulin genes is a strong prognostic predictor and BCR signaling has been postulated to have a role for CLL trafficking and interaction with the stromal microenvironment. There is also important evidence, favoring a role for the microenvironment in CLL pathogenesis. Most, if not all, proliferative events occur in the lymph nodes and bone marrow, where leukemic cells receive through microenvironment interactions survival signals aiming to avoid apoptosis and acquire favorable tumoral growing conditions. In addition, the tumoral microenvironment appears to be the site where the acquisition of additional genetic lesions in the clone occur, which should greatly influence clinical outcome. The advent of new tyrosine kinase inhibitors which seem to be able to modulate microenvironment interactions and circumvent the p53 deletion have generated significant promise by raising the possibility that they could provide significant progress in disease treatment. Most tumor cells are arrested in cell cycle G0/G1 stages and only a minimal portion of the clone displays a proliferative activity.2 Available treatments often induce remissions, although almost all patients relapse, and CLL remains an incurable disease.Chronic lymphocytic leukemia (CLL) is the commonest form of leukemia in Western countries and mainly affects elderly individuals. It follows an extremely variable course, with survival ranging from months to decades.3 Using a non-radioactive, stable isotopic labeling method to measure CLL kinetics, Messmer et al.4 demonstrated that B-CLL is not a static disease that results simply from the accumulation of long-lived lymphocytes, but a disease in which coexist a proliferative and an accumulative pool. In these conditions, the evolution of the disease probably depends on the relative importance of these pools.Recently, significant progress has been made in our understanding of CLL pathogenesis. CLL has traditionally been considered as an accumulative disease deriving from an inherent defect in apoptosis or in their cell death program. However, recent studies showed that B-CLL is a dynamic process composed of cells that also proliferate and die, often at appreciable levels.Important advances in the molecular pathogenesis were obtained through the study of the role of the B-cell receptor (BCR) and the microenvironment. In this review, we have tried to discuss, on the basis of our previous work and available evidence the role of the BCR and microenvironment in CLL evolution.6 In addition, it constitutes a foundational event in CLL ethiopathogenesis, as suggested by the fact that 20% of CLL cases from unrelated patients display extremely similar, sometimes even identical antigen receptors. In addition, BCR signaling has been postulated to have a role for CLL trafficking and interaction with the stromal microenvironment.7 However, since our initial publication,8 where we succeeded to show that there was a 10-fold decrease in the expression of membrane Igs, we recently could demonstrate that this poor expression was the consequence of an important defect in the assembly of the BCR.9 In addition, it is clear that when stimulated through the BCR pathway, the response of CLL cells is impaired. Low expression of the BCR correlates with reduced induction of protein tyrosine kinase activity and defective intracellular calcium mobilization and tyrosine phosphorylation.The BCR is an essential signal transduction pathway for the survival and proliferation of mature B lymphocytes and likely has a major role in the context of prognosis and positive selection of the precursor tumoral cell. Evidence for the important role of the BCR in CLL pathogenesis is given by the fact that the mutational status observed in the BCR sequences is one of the strongest predictors of disease outcome.10Although the possibility that BCR signaling has an important role in CLL evolution and progression, although this possibility cannot be excluded, concerns can be raised. Available evidence exists suggesting that the interaction of leukemic cells with stromal and T cells in the microenvironment has a key role in CLL evolution. It is clear that most, if not all, proliferative events occur in the lymph nodes (LN) and bone marrow, where leukemic cells are able to exploit microenvironment interactions with stromal cells and T-lymphocytes, in order to avoid apoptosis and acquire favorable tumoral growing conditions. As there is increasing evidence that individual cancer samples are heterogeneous and include subclonal populations and that tumors likely evolve through competition and interaction between different subclones, this tumoral microenvironment appears to be the site where acquisition of additional genetic lesions in the clone occur, which, should greatly influence clinical outcome. In line with this hypothesis, the LN CLL B-cells were found to show increased proliferation when compared with peripheral blood (PB) cells and were found to have a gene expression profile compatible with activated B cells.11 and Bruton tyrosine kinase (BTK).12 These drugs share a unique pattern of response resulting in nodal reduction and increased PB lymphocytosis. This likely reflects microenvironment modulation, which could lead proliferative leukemic cells, by an, as yet, unknown mechanism, to abandon protective microenvironment niches and thus induce them into apoptosis. In addition, these drugs are better tolerated than traditional chemotherapies and might be efficacious in the setting of traditional high-risk prognostic factors such del(17p). Thus, these new drugs targeting the signalosome have generated considerable excitement and could have a major role in new treatment strategies aiming at curing disease, as they could target the proliferative pool.During recent years, a variety of novel kinase inhibitors aiming to target various components of the BCR signaling pathway have been designed. These mainly include phosphoinoside 3́kinase (PI3K)8 The BCR pathway is used by normal B cells to promote cell proliferation and to induce antibody production. Once encountering the antigen, the activated BCR recruits kinases such as spleen tyrosine kinase (SYK) and LYN, which phosphorylate the immunoreceptor tyrosine-based motifs receptors of Igα/Igβ.13 Phosphorylation of these induces a cascade of downstream events, which include that activation of BTK and PI3K,14 which subsequently induce the mobilization and activation of other downstream kinases such as protein kinase C-β, mammalian target of rapamycin and mitogen-activated protein kinase ERK.15 The activation of this cascade promotes survival and proliferation of B cells through the upregulation of transcription factors such as nuclear factor κ β (NF-κβ).16The BCR is a multimeric complex formed by the assembly of surface immunoglobulin (Ig) homodimer and the noncovalently bound heterodimer Igα/Igβ (CD79a/CD79b). This latter has a key role for receptor expression and signal transduction, by linking the antigen-binding Ig chains to intracellular tyrosine kinases of the Src-family.17The BCR is critical for normal B lymphocyte survival and is retained in the majority of B-cell malignancies. Thus, it constitutes a privileged marker as the BCR has unique Ig rearrangements that characterize the tumor cell and reveal the nature of its B-cell origin. Three main phenotypic features define the CLL B cell: (a) the predominant population shares B-cell markers  with the CD5 antigen, in the absence of other pan-T-cell markers; (b) the B cells are monoclonal with regard to expression of either κ or λ light chains; and (c) express surface immunoglobulin, CD79β, CD20 and CD22 with low density. These characteristics are generally adequate for a precise diagnosis of CLL, and they also distinguish CLL from other disorders such as prolymphocytic leukemia, hairy-cell leukemia, mantle-cell lymphoma and other lymphomas.18 there is consensus on the absence of genetic defects in the BCR components.19 Although there are individual differences among patients, almost all CLL cases display very low levels of surface IgM and CD79β, when compared with normal B cells. In contrast with their poor expression at the membrane level, the mRNA transcription as well as intracellular synthesis of BCR components appear to be normal21  where proteins are modified  and then properly folded before exit to the Golgi. If the folding and maturation process fails, several different mechanisms are used by the quality control system of the cell to avoid the production of non-native proteins. The proteins are retained in the ER and exposed to resident chaperones to attempt adequate folding. In the case of BCR, several chaperones such as calnexin, calreticulin, glucose-regulated protein 78 also known as BiP (Ig heavy chain-binding protein) and GRP94 have been shown to associate with μ CD79α and CD79β chains.23 we found that nascent IgM failed to be processed and transported to the membrane. To gain insight into the relationship between this defect and IgM surface expression, we analyzed the different components of the BCR for their glycosylation status in 10 CLL patients. Six CLL cases displaying very low expression (patients no. 1–6), and higher, although significantly lower than normal, for patients no. 7–10 individual differences concerning surface IgM expression. In a previous work,9 With normal cell extract, only a minority of μ and CD79α chains shifted to, respectively, 70 kDa and 25 kDa bands corresponding to immature glycoforms  from Golgi-processed (mature) glycoforms.As a consequence of their glycosylation defect and inability to assemble, cytoplasmic BCR components are retained in the ER compartment by the chaperone protein calnexin and cannot be transported from to the cell surface. 24 Nevertheless, as the membrane expression of surface immunoglobulin is very poor in CLL, this only concerns a minority of the expressed intracellular Igs.A recent study, confined to surface Ig confirmed this glycosylation defect and demonstrated that a percentage of immature glycoforms could, however, reach the surface and postulated that these immature Ig glycoforms that predominate among UM cases could provide tonic survival signals.17 However, 50–70% of CLL harbor somatic mutations of IgVH genes25 as if they had matured in a lymphoid follicle. Although the origins of the CLL leukemic clone remains unsolved,27 the presence or absence of somatic mutations is associated with the use of particular IgVH genes.28 Two reports demonstrated that the clinical behavior of CLL is related to the mutational status of Ig genes. Patients whose B cells express mutated VH genes have a more indolent disease and longer overall survival than do patients expressing UM genes.6 The mutational profile of Ig genes delineates prognostic groups within all Binet's stages.1 Despite this important difference in terms of prognosis gene expression profiling identified mutated and UM CLLs as part of a shared disease process with a common gene expression signature.30Most B-CLL cells express CD5 and IgM/IgD and thus have a mantle zone-like phenotype of naive cells, which, in normal conditions express UM Ig genes.31 In recent years, several groups have reported differences in IgVH gene usage in CLL among different geographic areas .32 Also, somatic mutations are not uniformly distributed within IgVH gene families, as they predominate among IgVH3 and IgVH4 families, whereas an UM profile is prevalent in the IgVH1 family. The most frequently used IgVH genes in CLL rearrangements in Western countries are IGHV1–69, IGHV3–07, IgVH 3–21 and IgVH 4–34.32 However, the available data from Japan, China and Iran show some differences with those from Western countries. The frequency of VH1–69 in Japan and Iran is lower than that in Western countries. In addition, the frequency of the VH 3–21 gene usage appears to be higher in Northern European countries when compared with Mediterranean ones. Evidence for the notion that CLL is a tumor of antigen-experienced B cells comes from the structure of the rearranged IgV genes. Analyses of large panels of CLL cases revealed that certain IgV gene family members, which could be hypermutated or UM, were expressed significantly more frequently in CLL than would be expected from their expression in the IgV gene repertoire of normal B cells.28 Although there is evidence favoring the idea that BCR stimulation by the antigen could have an important role in CLL evolution, concerns have been raised against this possibility. To formally prove this hypothesis, we should be able to proceed to stimulation of CLL cells with the antigen recognized by the BCR. Unfortunately, this putative antigen remains unknown.The analysis of IgVH repertoire in CLL has demonstrated biases both at the family and specific gene segment usage.33 Given the key role of this protein in cell cycle progression, its overexpression in CLL cells may account for the accumulation of B cells in early phases of the cell cycle.17 In addition, overexpression of the anti-apoptotic BCL-2, BCL-XL, BAG-1 and MCL-1 molecules and the absence of microRNAs miR-15 and miR-16,34 whereas proapoptotic proteins like BAX and BCL-XS are under expressed35 could explain the resistance of tumoral cells to apoptosis. Despite the fact that most leukemic cells are arrested in cell cycle G0/G1 stages, Messmer et al.4 demonstrated that CLL is not only a static disease but also a disease in which a proliferative pool coexist. In consequence, evolution of this leukemia could depend on the relative balance between these subpopulations.CLL can be defined as a low-grade CD5+ B-cell tumor, whose tumoral cells have previously encountered the antigen, escaped programmed cell death and undergone cell cycle arrest in the G0/G1 phase. In CLL cells, elevated levels of the cyclin-negative regulator p27-Kip1 protein are found in a majority of patients.in vivo results, apoptosis occurs after in vitro culture, suggesting a role of the microenvironment in CLL cell survival.36 Within the leukemic microenvironment, two cellular components appear to be potential players: stromal cells and T-lymphocytes. In vitro, the spontaneous apoptosis of B-CLL can be rescued by stimulation via surface CD40 and interleukin-4,37 by the coculture with mesenchymal stromal cells38 and/or ‘nurse-like cells'.39In contrast with 4041 and thereby create a microenvironment that supports their own survival. There is an increase of CD3+ T cells, most of which are CD40L+CD4+, which cluster in and around pseudofollicles. These cells can stimulate CLL cells through the interaction of CD40 and CD40L, and this stimulus synergizes with BCR signaling.42 In addition, evidences exist that CD40–CD40L engagement activates NF-κβ/Rel transcription factors as well as Janus-activated kinase (JAK 3) and signal transducer and activator of transcription 3 to induce high levels of the anti-apoptotic proteins BCL-XL and MCL-1.43 NF-κβ activation, in turn, leads to upregulation of the TP63 which also acts through the integrin very late antigen-4 (CD49d) to facilitate the migration of CLL cells into the supportive microenvironments.44 When cocultured with CLL cells, mesenchymal stromal cells appears to be able to protect the neoplastic B cells from apoptosis induced by fludarabine and bendamustine.45 In turn, nurse-like cells are also able to protect CLL cells from apoptosis.46 Nurse-like cells secrete stromal cell-derived factor 1 and the tumor necrosis factor family ligands APRIL (a proliferation-inducing ligand) and BAFF (B-cell-activating factor), which protect CLL cells from apoptosis.47 Both mesenchymal stromal cells and nurse-like cells therefore have anti-apoptotic activity and the combination of these two cell types provides a supportive microenvironment for tumor cells in CLL.48CLL cells seem to recruit accessory cells;49 and CXCR4 and CXCR5, which are differentially expressed by the B cells.50 In addition, bone marrow and LN stromal cells secrete CXCL9,10,11,12,13 and CCL19 and 21 which bind CXCR3, CXCR4 and CXCR5 receptors different expressed by the leukemic clone.51 In turn, CLL B cells, also secrete chemokines such as CCL3, CCL4, CCL22 and interleukin-8, which can attract accessory cells, such as T cells and monocytes. This finding suggests that CLL cells are not simply seed in a supportive soil but instead are actively involved in a complex crosstalk that possibly will maintains a proliferative behavior.Lymphocyte trafficking between the blood and secondary lymphoid tissues is organized by tissue-specific expression of chemokines and their receptors on lymphocytes, which cooperate with adhesion molecules and their ligands. B-cell positioning in these areas is coordinated by the chemokines CXCL12 and CXL13, which are expressed by stromal cells,52 Our results, demonstrated that this aberrant expression resulted from the lack of methylation in the lipoprotein lipase CpG island. Interestingly, this epigenetic mechanism appeared to be mainly triggered by T-cell-dependent microenvironments signals (CD40L+interleukin-4), whereas cross-linking of the BCR poorly induced this demethylation process, and signaling through TLR9 or TLR1/2 pathways were found unable to induce it.Evidence indicates that the relationship of CLL B cells with their microenvironment is crucial for the creation of altered key proliferative/apoptotic pathways, which are hallmarks features in CLL pathogenesis. In a recent work, we have investigated the mechanisms accounting for the aberrant expression of the lipoprotein lipase enzyme in UM CLL patients.41 Therefore, understanding the crosstalk between malignant B-cells and their milieu could give us new keys on the cellular and molecular biology of CLL that can finally lead to novel strategies for disease treatment. In this regard, the isolation and analysis of the tumoral subset that is being triggered in the proliferative compartments of progressive CLL cases is an important aim to understand CLL pathogenesis. Different groups have tried to assess this question by studying different CLL proliferative subsets like that expressing CD38 marker,53 or that expressing activation induced cytidine deaminase (AID)54 or CD5/CXCL4 molecules.55At this time, it is clear that crosstalk with accessory cells in specialized tissue microenvironments favors disease progression by promoting malignant B-cell growth and the emergence of new genetic alterations, which will lead to drug resistance.56 AID is expressed mainly in stimulated B cells and at the physiological levels is responsible for DNA damage in the pre-switch and variable VDJ regions that lead to DNA breaks and/or point mutations in the immunoglobulin genes.Under physiological conditions, the microenvironment stimulation occurs in the germinal centres and is triggered by the presence of an antigen. In this microenvironment, B lymphocytes undergo a series of specialized events resulting in the production of specific antibodies with both high affinity and adequate effector function. These processes are somatic hypermutation, in which different mutations are introduced into the variable domain of Igs and class switch recombination (CSR), which results in a change of antibody isotype. A useful humoral immunity is dependent on these two processes and both are absolutely dependent on AID enzyme.57 although this expression is confined to a small proportion of the CLL clone.58 As AID expression results from signals mainly received through the CD40–CD40L pathway, we subsequently investigated whether its expression was related to CSR occurring in a small subset of CLL B-cells and whether it could have an increased proliferative potential. Our results showed that high AID expression is almost exclusively restricted to the subpopulation of tumoral cells having an ongoing CSR process and more important, the presence of this subpopulation in CLL is closely related to an aggressive course of the disease.59 This small clonal subset having achieved CSR and expressing either IgG at the membrane or both IgM and IgG, displays higher levels of AID, and higher levels of proliferation and anti-apoptotic molecules like Ki-67, c-myc and Bcl-2. In addition, it also expresses high levels of proteins associated to progression such as CD49d and CCL3/CCL4 chemokines as well as a decreased expression of the cell cycle inhibitor p27-kip1 when compared with its quiescent tumoral counterpart expressing exclusively IgM  and APRIL (proliferation-inducing ligand)47 or TLRs (Toll-like receptors)63 and TLRs and BCR64 are also able to trigger AID expression and CSR. Thus, ongoing CSR and AID expression in the PB of UM progressive CLL cases appears to be a hallmark of a proliferative disease in which B lymphocytes are being constitutively activated in specific tumor microenvironments.65It is accepted that AID expression is induced in germinal centres through the contact of T and B cells via CD40–CD40L interactions.10 CLL cells in the LN showed upregulation of gene signatures indicating BCR and NF-κβ activation. In addition, the expression of these genes was stronger in clinically more aggressive CLL and tumor proliferation as assessed by E2F, and c-MYC and Ki-67 expression was higher in LN and was correlated to disease progression. The authors concluded that these results could be useful to identify target molecules able to disrupt the tumoral microenvironment interactions and to inhibit the BCR signaling as promising therapeutic strategies in CLL.1066A recent study compared gene expression profiling of PB to LN and bone marrow tumoral cells in 24 treatment naïve patients. This study identified LN as a key site for proliferation.et al.10 in LN from CLL patients. This small subpopulation expressing AID in the PB of progressive UM CLL patients, probably results from the passage into the circulation of this proliferative pool occurring in LN. In agreement with this hypothesis, this pool can be identified in the PB of some UM CLL patients displaying a highly aggressive disease ,61 it is logical to assume that progressive disease could be related to clonal CLL evolution. In this regard, it has been also suggested that mutations in TP53 genes could be related with AID expression in CLL.67 If true, the constitutive expression of AID in the leukemic clone history could be a key event in disease progression.Being aware that AID overexpression is associated with the loss of target specificity resulting in mutations in non-immunoglobulin genes, , which exhibit a low response, appear to respond better than mutated ones.CLL is characterized by an accumulation of monoclonal B lymphocytes expressing CD5 and CD23 molecules, but critical low amounts of surface Ig and CD79β molecules.Normal B cells following interaction with antigen-specific T cells or not in the case of T-independent responses should differentiate into plasma cells or memory cells. Those cells failing to be selected by their antigen affinity should be committed to death. In CLL, the tumoral process protects these cells from following apoptosis and allow them to survive without differentiation.The BCR is an essential signal transduction pathway for the survival and proliferation of mature B lymphocytes and likely has a major role in the context of positive selection of the precursor tumoral cell. Thus, it constitutes a foundational event in CLL ethiopathogenesis and as suggested by the fact that 20% of CLL cases from unrelated patients can have extremely similar, sometimes even identical antigen receptors, this probably occurs as a consequence of a stringent selective process induced by the antigen. Antigen likely has an important role at one point during the ontogeny of CLL in the context of positive selection of the precursor cell. It is presently unclear whether it has a major role during the evolution of disease, as shown in other tumoral models like mouse lymphoma models or in some mucosa-associated lymphoid tissue lymphomas, where continuous antigen stimulation has a key role and antigen withdrawal leads to regression of disease.in vivo which could induce signaling events for constitutive activation of kinases and of NF-κβ in CLL B cells.During recent years, evidence accumulated suggesting that signaling via the BCR could have an important role in the development of CLL and that it could determine the variable clinical behavior of the disease. This could likely occur through antigen exposure 68 These drugs share a unique pattern of response resulting in nodal reduction and increased PB lymphocytosis likely reflecting that microenvironment modulation, which, could prevent these cells to receive the survival signals delivered by the microenvironment.The advent of new therapies that target the signalosome brings new evidence in favor of this view. Initial results from clinical trials with drugs that inhibit SYK kinase (fostamatimib), the BTK kinase  and PI3K-δ  are very encouraging.In addition, these drugs are better tolerated than traditional chemotherapies and might be efficacious in the setting of traditional high-risk prognostic factors such as del(17p). Thus, these new molecules targeting the ‘signalosome' have generated considerable excitement and could have a major role in new treatment strategies aiming at curing disease, as they could target the proliferative pool.23 that transcription and intracellular synthesis of the BCR components were normal and that low BCR expression at the membrane resulted as a consequence of defective assembly of the different BCR components. Our results showed a severe impairment in glycosylation and folding of μ and CD79α chains, leading to their defective assembly and their retention in the ER compartment, which could account for the low levels of IgM surface expression in malignant B-CLL cells. Although a recent study confined to membrane Igs demonstrated that some of these immature glycoforms succeed to assemble and reach the membrane, the amount of Ig and particularly CD79α and CD79β that normally reach the membrane is very low and the majority of Ig produced by the cell remains retained in the reticulum endoplasmic compartment.We previously describedMoreover, even if we accept that UM cases respond better to stimulation through the BCR than the mutated ones, the response is poor in the vast majority of cases and at least one-third of these cases display very low responses to this stimulation.64 Thus, the contribution of specific BCR signaling inhibition to the clinical efficacy of these agents remains unclear as yet. Indeed, it is possible that their clinical effects stem from simultaneous inhibition of multiple signaling responses.Although promising, it may be premature to ascribe positive clinical responses of SYK, BTK and PI3k-δ inhibitors to specific inhibition of BCR-mediated signaling. In particular, these kinases also contribute to other signaling pathways independent of the BCR. For instance, in CLL, SYK phosphorylation is increased in cells stimulated via chemokine receptors and integrins and SYK inhibition reduced migration toward CXCL12 and adhesion to VCAM-1. The PI3K inhibitor CAL-101 also interferes with survival effects of CD40L, tumor necrosis factor-α and fibronectin in CLL cells and BTK is required for Toll-like receptor signaling.et al.10 in the LN, we can speculate that these cells result from the passage into the circulation of the proliferative pool occurring in the LN.There is increasing evidence that individual cancer samples are heterogeneous and include subclonal populations and that tumors likely evolve through competition and interaction between different subclones. In the case of CLL, this so called proliferative pool may be the site where acquisition of additional genetic lesions in the clone occur. AID expression is induced through the contact of T and B cells via CD40–CD40L and other interactions, which results from signals received in this particular microenvironment. Our data indicate that the proliferative subset expressing AID and proliferative markers in PB, is the consequence of microenvironment interactions occurring in the specific proliferative CLL centres. As this subset expresses very similar markers to those found by Herishanu Overall, these new approaches have allowed better understanding of CLL pathophysiology and provided a more effective and rationale criteria for the management of this disease. How do these data help defining treatment in CLL?Current chemotherapy options are able to obtain complete and long remissions, although relapse ineluctably occurs and a second round of chemotherapy usually results in inferior response. This probably results because we are presently unable to eliminate residual malignant clones located at the LN and bone marrow, which through their interaction with stromal and T cells, receive survival signals and are able to produce new genetic lesions like the 17p or 11q deletion.Recent results pointing to an important role of BCR and microenvironment signaling in tumor progression may redirect therapeutic options to treatment aiming to selectively attack the proliferative pool existing in this protective microenvironment.


Health information exchange and health information integration has become one of the top priorities for healthcare systems across institutions and hospitals. Most organizations and establishments implement health information exchange and integration in order to support meaningful information retrieval among their disparate healthcare systems. The challenges that prevent efficient health information integration for heterogeneous data sources are the lack of a common standard to support mapping across distributed data sources and the numerous and diverse healthcare domains. Health Level Seven (HL7) is a standards development organization which creates standards, but is itself not the standard. They create the Reference Information Model. RIM is developed by HL7's technical committees. It is a standardized abstract representation of HL7 data across all the domains of health care. In this article, we aim to present a design and a prototype implementation of HL7 v3-RIM mapping for information integration of distributed clinical data sources. The implementation enables the user to retrieve and search information that has been integrated using HL7 v3-RIM technology from disparate health care systems.We designed and developed a prototype implementation of HL7 v3-RIM mapping function to integrate distributed clinical data sources using R-MIM classes from HL7 v3-RIM as a global view along with a collaborative centralized web-based mapping tool to tackle the evolution of both global and local schemas. Our prototype was implemented and integrated with a Clinical Database management Systems CDMS as a plug-in module. We tested the prototype system with some use case scenarios for distributed clinical data sources across several legacy CDMS. The results have been effective in improving information delivery, completing tasks that would have been otherwise difficult to accomplish, and reducing the time required to finish tasks which are used in collaborative information retrieval and sharing with other systems.We created a prototype implementation of HL7 v3-RIM mapping for information integration between distributed clinical data sources to promote collaborative healthcare and translational research. The prototype has effectively and efficiently ensured the accuracy of the information and knowledge extractions for systems that have been integrated Health information integration (HII) is one of the crucial challenges for healthcare systems to integrate heterogeneous data sources into a standard format. This process allows researchers to extract information and knowledge from the integrated systems effectively and efficiently using information retrieval (IR) methods, such as semantic similarity mapping . Many prWith the use of well defined and widely accepted standards, information can be integrated and shared within and across healthcare domains enabling HIE to be implemented. With the vision of an integrated system, there should be only one integrated schema  that combines all distributed data sources across several disparate systems and allows the integrated system to seamlessly and efficiently extract relevant information and evaluate the effectiveness of HIE. Various information integration techniques have been proposed in the past, for example schema integration ,4, data Srikrishnan et. al. proposed an integrated system from distributed databases using the hyper-graph data model which represents database schema in a low-level data model . SeveralExisting legacy clinical data management System (CDMS) refers the system that is in used before the introduction of HL7 based system (v2 or v3), while all references to "HL7 legacy systems" refer to HL7 v2 implementations since v2 is the older of the two technologies. A comparison of HL7 v2 and v3 messages reveals that they are incompatible with each other, as their message formats are completely different. v2 messages use delimiters ("|" symbol) to separate values, whereas v3 messages use Extensible Markup Language (XML) data objects for values. Also, HL7 v2.3.1 and later versions support XML encoding of messages, but the HL7 v3 XML specification is built around vocabularies and data types appropriately extracted from RIM ,15. v3 wScientists and researchers have been using one technique or the other in order to integrate their CDMS legacy systems and HL7 v2 systems with the HL7 v3 system. In an effort to build a unified format for a National Information System in Turkey, The Ministry of Health implemented the Clinical Document Architecture Release Two (CDA R2) standard and a client application compatible with HL7 v3 for information interchange . Yang etIn this article, we developed a prototype to integrate distributed clinical data sources using R-MIM classes from HL7 v3-RIM as a global view along with a collaborative centralized web-based mapping tool to tackle the evolution of both global and local schemas. Our prototype was implemented and integrated with a clinical data management system as a Plug-in module using a CDMS. A clinical data management system  is uThe HL7 technical committees developed the HL7 v3 message structure that is based on the reference information model (RIM). The objective of this model is to tackle the bottleneck involved in the information interchange among health information systems. The RIM is the data source, with a coherent and shared information model that is necessary for the data content of all the HL7 v3 messages. The RIM is an abstract model-driven development methodology based on a unified modelling language (UML) and the root of all information models that represent the HL7 data in a standard way across all the domains of healthcare system. It is also a complete HL7 v3 reference model that includes all the object attributes and properties and state transition diagrams that specify the life cycles of all class objects . For speRIM core classes contain the following subject areas: Acts, Entities and Roles. The Acts subject area contains the following classes: Account, Act, ActRelationship, ControlAct, Device Task, DiagnosticImage, Diet, FinancialContract, FinancialTransaction, InvoiceElement, ManagedParticipation, Observation, Participation, PatientEncounter, Procedure, PublicHealthCase, SubstanceAdministration, Supply and WorkingList. All the classes in the Acts subject area relates to all the events and actions in the health care services . The EntOther subclasses can be derived or cloned from the core classes . The clone classes can be viewed as a direct or conceptual specialization of the core class. There are invariably thousands of clone classes with the core or specific domain classes of RIM .Once the domain is specified, a Refined-Message Information Model (R-MIM) is used to express the content for a set of messages with incumbent annotations and elaborations that are message specific. To exchange information between systems, the Hierarchical Message Descriptions (HMDs) represents the message structures or message types that are used to express R-MIM abstract message structures in an organized way, which can be communicated between systems with disparate underlying technologies. One of the most important things the HMD does is to specify the serialization of the two dimensional R-MIM into a one dimensional data stream. It also involves the Implementation Technology Specification (ITS) through Extensible Markup Language (XML) and Unified Modeling Language (UML) ,26. FurtThe HL7 data types and structures are defined by XML ITS. It follows the extendable markup language protocols, while the structures represent the constructs defined by HMDs. Thus for every HL7 message type it is necessary to have an HMD and XML Schema Definition (XSD) to express a set of rules to which XML document structure must follow in order to be considered valid according to the schema specifications. Thus the XSDs contain all required information that is essential for constructing a complete HL7 v3 message.An understanding of the artifacts for domain-specific models is essential for understanding the HL7 v3 specification. For every domain, the artifacts are organized in the same structure that is submitted by the HL7 technical committee during the specification development process. For instance, an application role submitted by the Patient Administration under Administrative Management Domain will have a unique artifact identifier like PRPA_ IN101001UV 01 where PR = Practice (Subsection), PA = Patient Administration (Domain), IN = Interaction (Artifact type), 101001 = 6 digit non-meaningful number assigned by the Technical Committee to ensure uniqueness, UV = Realm , and 01 = Current version number. The root element uniquely identifies the message's interaction identifier, which identifies the message type, the trigger event, and the receiver responsibilities. In this example, the interaction between two systems is defined by the interaction code (IN). The IN shown in the actual message would be composed of the Trigger Event, the Message Type, the Transmission Wrapper, the Control Act Wrapper, the Sender, and the Receiver. The Trigger Event and Control Act Wrapper represent another wrapper around the actual message, which explains information about the date and time the trigger event occurred as well as the responsible parties for the trigger. The development of RIM opens the door to significant movement from HIE standards through messaging to an integrated healthcare systems architectural paradigm. RIM represents all the attributes and data elements that are needed for HL7 message communication and data exchange ,25.Compared with other implementations of Health Information exchange ,10,12,13The integrated system we developed consists of several key functions, as shown in Figure The key functions of integration workflow are listed:• Administrator's User Interface (UI) - Administrator interface provides the user an interface to submit queries to the integrated system .• SQL optimizer - The submitted queries are used in close collaboration with global view based on HL7 v3-RIM to decompose the query into subqueries which efficiently and effectively follows the stated plan usage to optimize the execution plan for query processing time.• Global View based HL7 v3-RIM - The R-MIM class represents domain specific healthcare model.• Relevant Data Extraction - The sub-queries are sent to appropriate distributed legacy CDMS through this function. The partial results are effectively and efficiently combined to finalize the integrated result.• Web-based Mapping Tool - The online centralized web-based mapping tool allows multiple sites in a clinical trial study to access and perform the mapping between each legacy CDMS and R-MIM classes (See 3.1).• Legacy to HL7 RIM Mapping Information - The Oracle database engine for data storage and manipulation for legacy CDMS to HL7 RIM mapping information is securely housed in CDMS Figure .Once the mapping information is created, the administrator can perform the query against the global view through the UI that has been provided. By consulting through the mapping information, the query is decomposed into subqueries and processed against the global view HL7 v3-RIM). The partial result extractions are retrieved from each site and combined to form the final result . The HL7 Java SIG API (Java SIG group is now the RIM Based Application Architectures (RIMBAA) work group) provides an open source Java Application Program Interface (API) that implements the definition of HL7 v3 RIM . The OpeAn HL7 v3 is said to have used RIMBAA technology if at least one of the nine cells is used in message/document transfer or in other RIM-based functions -32. RP, https://sourceforge.net/projects/hl7mtsdemo/. Figure The HL7 v3 message has been integrated with CDBMS. We implemented a drug administration use case (PORR_MT040002) using clinical trials in three data sources with different underlying database technologies. HL7 v3 messages are generated Figure  Figure 6The HL7 v3-RIM development has opened significant movement to support HII and HIE across nationwide healthcare community. However, adopting RIM standard requires a thorough and satisfactory understanding of each and every concept involved. The development of such automation will facilitate mapping between underlying healthcare CDMS and R-MIM and can be used in addressing disparate healthcare system integration issues. The automated mapping service that uses widely accepted methods like pattern matching, semantic mapping and machine learning techniques will enable the mapping process to be dynamic and support real-time collaborative information sharing across translational research community. In addition, this service would minimize the need for manual tracing of all attributes in RIM, which is a difficult and a time-consuming task.The automated mapping engine is being built on top of the open source Apache Lucene information retrieval library to partially automate the mapping of the legacy database constructs into the relevant D-MIM components. Lucene library provides a rich query language through the query parser that uses JavaCC by Sun Microsystems. Once this process is complete, it will provide term searches, fuzzy searches based on the Levenshtein Distance or Edit Distance algorithm to Proximity ,33,34. TClinical Document Architecture (CDA) is a clinical exchange document authored by HL7. It is based on XML markup structure and used in specifying the standard in terms of syntax, structure and semantics. It is used in creating the HL7 RIM and uses the HL7 v3 data types -25. It iContinuity of Care Record (CCR) is a health information technology exchange standard that is designed, developed and maintained by group of volunteer healthcare information technology professionals under the leadership of ASTM International. It is formally referred to as "E2369-06". CCR contains a digital summary or snapshot of the most relevant of a patient's administrative and clinical health information which can be transmitted or transferred between disparate healthcare systems. It is used in moving medical/health record among facilities (inter-facility movement). It is also based only on XML object oriented relational models ,36.HL7 v3 is based on the reference information model (RIM) which has the objective to tackle all the bottleneck involved in the information interchange among health information systems, thus HL7 v3 is used in moving medical/health care records among healthcare facilities (intra-facility movement). The RIM is an abstract model-driven development methodology based on a unified modelling language (UML) and the root of all information models that represent the HL7 data in a standard way across all the domains of healthcare systems. HL7 v3 also uses the XML data model which includes both data and metadata in a unified format. Also HL7 v3 is triggered as a real-time protocol while CCR is not triggered as a real-time protocol ,36.Compared with other implementations of health information exchange ,10,12,13To assist physicians and other healthcare providers in undertaking and preparing a nationwide health information integration (HII) and health information exchange (HIE) standards, we have made substantial progress, most notably through the creation of a prototype implementation of HL7 v3-RIM mapping for information integration between distributed clinical data sources to promote collaborative healthcare and translational research. Although understanding RIM is necessary for implementing HL7 v3 message standards to solve today's complex healthcare information interchange requirements, the complex standard covers various health information domains extensively and provides a common acceptable standard for HIE. Thus, adopting such standard as a global view will be significant progress in the integration of data from disparate CDMS. We have tested several sample queries against the clinical data integration workflow using the prototype. The approach has effectively and efficiently ensured the correctness of the information and knowledge extractions for systems that have been integrated. In the future we intend to implement the results of the subqueries from the disparate systems that are integrated into a final solution query solution.The authors declare that they have no competing interests.All the authors contributed equally to this work. All authors read and approved the final manuscript


Decompressive hemicraniectomy is a life-saving procedure for patients with malignant middle cerebral artery infarctions. However, the neuropsychological sequelae in such patients have up to now received little attention. In this study we not only describe neuropsychological deficits but also the quality of life and the extent of depression and other psychiatric symptoms in patients after complete media infarction of the non-speech dominant hemisphere.20 patients from two different university hospitals (mean ± standard deviation: 52 ± 14 years of age) who had undergone hemicraniectomy with duraplasty above the non-speech dominant hemisphere at least one year previously were examined using a thorough neurological and neuropsychological work-up. The quality of life and the extent of psychiatric problems were determined on the basis of self-estimation questionnaires. The patients were asked whether they would again opt for the surgical treatment when considering their own outcome. 20 healthy persons matched for age, gender and education served as a control group.All patients but one were neurologically handicapped, half of them severely. Age was significantly correlated with poorer values on the Rankin scale and Barthel index. All cognitive domain z values were significantly lower than in the control group. Upon re-examination, 18 of 20 patients were found to be cognitively impaired to a degree that fulfilled the formal DSM IV criteria for dementia.Patients with non-speech dominant hemispheric infarctions and decompressive hemicraniectomy are at high risk of depression and severe cognitive impairment. For more than a century, trephining of the skull to relieve intracranial pressure due to brain swelling has been a treatment option for space-occupying lesions such as large brain infarctions in the area supplied by the middle cerebral artery (MCA) . With moBetween 2004 and 2008, we contacted patients who had suffered from malignant infarction of the MCA and had been treated using DCH with duraplasty at a university medical center  between 1999 and 2003. A total of 58 patients were admitted to the Göttingen university clinic for malignant MCA infarctions during this time. In 1999, there were 18 patients, all of whom had been treated conservatively. Between 2000 and 2003, 40 patients with malignant MCA infarction were admitted, 19 of whom had been trephined and 21 treated conservatively. From 2000 on, patients with malignant MCA infarctions were also treated using DCH. All operated patients had infarctions of the non-speech dominant hemisphere. Seventeen of the operated patients could be re-examined; only one patient could not be located. Of the 39 conservatively treated patients, 18 died while still hospitalized. Seven of the conservatively treated patients could not be found for re-examination. In addition, we examined the 11 surviving patients with non-speech dominant hemispheric infarctions of the Jena University Hospital who comprised the patient population reported by S. Harscher  21.8 [15.6/52.5]). Eleven of the patients had undergone DCH in the early course (< 24 h after onset of symptoms) and in 9 patients, DCH had been delayed.The patient group (P) and the group of 20 healthy control persons (C) were matched for age (mean ± SD 55.5 ± 13.7th/75th] 2.4 [1.7/3.5]) years later.Re-examination of patients was carried out at a mean ± SD 3.0 ± 0.8 . Upon re-examination of the patients who received DCH, 17 patients lived at home; three were living in a nursing home. Nine of 20 patients had developed symptomatic epilepsy, and 13 had been diagnosed with post-stroke depression; 11 of them were treated at the time of examination with anti-depressive agents. Confronted with the hypothetical situation of turning back time to the decision to undergo DCH, the majority of patients stated that they would again opt for a decompressive hemicraniectomy , and the median [25th/75th percentile] was 46.5 [17.5/76.5]. The median modified Rankin scale was 4 , and 5 patients had a mRS < 4 while 15 patients were affected severely (mRS 4 and 5).At the time of re-examination the median  z value -3 [-3/-1.2]P vs. -0.02 [-0.4/0.77]C, p < 0.001). Thirteen of 20 patients after DCH were depressive (compared to none in the control group). Of our patients, only half were treated with anti-depressive drugs. No patient underwent psychotherapy. BDI z values from patients without epilepsy were not significantly lower than those of patients with epilepsy .The mental symptom load for all domains analyzed by SCL90r was higher in patients compared with healthy controls. These differences reached statistical significance for the domains "somatization", "obsessive-compulsive", "depression", "anxiety", "phobic anxiety", and "psychoticism". However, only for the subscale "depression" were these differences higher than one standard deviation. As compared to the control group, depressive mood represents the main mental problem in patients after DCH  but they were correlated significantly with the BDI z values . All WHOQoL subscales and the WHOQoL-Brefoverall score were positively correlated with each cognitive domain.In all domains the analysis of the WHOQoL Bref questionnaire showed results indicative of lower quality of life in patients compared with the control group Figure . Mean z That DCH can be a life-saving treatment option for complete MCA stroke is no longer a matter of debate -11,37-39The self-assessment of mood in our patient group revealed significant impairments. In contrast to Leonhardt , but conIn regard to cognitive outcome after DCH, our results may be too negative since 8 of our patients were older than 60 years, whereas other recent studies included only patients up to 60 years . FurtherThe higher age of our study population also explains the high proportion of patients with an unfavorable Rankin scale (75% in our study) compared to 43% in pooled data from the DESTINY, HAMLET and DECIMAL trials. Thus, the population analyzed here is certainly not comparable to the study groups in the above mentioned studies on DCH. The same is true for the variance of time from stroke onset to the neuropsychological tests. Therefore, our data should be interpreted cautiously with respect to these issues and might not be valid for a younger population examined within a narrower time interval after the treatment. Our findings are comparable to another study with a similar rate of unfavorable outcome (73.7%) [Another limitation of our investigative focus on cognitive deficits is that patients with speech-dominant hemispheric stroke were not included. Our findings for the depression questionnaires, therefore, are not transferable to patients with DCH over the speech-dominant hemisphere, as it is well known that mood disturbances appear more often in patients with right-sided stroke than in those with left-sided cerebral ischemia ,47. StraIn regard to both mood and cognition, our findings may significantly differ from patients with left-sided MCA infarctions and DCH. Since this study focused on cognitive functioning, reliable results were dependent on the patient's understanding of the test rules. The degree of comprehension is extremely difficult to quantify in partially aphasic patients, and there is no general rule as to how to correct the cognitive results for the individual type of aphasia. Therefore, we decided to exclude patients with speech-dominant hemispheric infarctions rather than misinterpreting their cognitive test results.As in other studies on DCH, most of our patients were able to walk after DCH and had regained some independence, despite the persisting physical handicaps . PhysicaThe presence of symptomatic epilepsy should have an influence on cognitive functioning as well as mood . ParticuAll told, the findings demonstrate that the probable outcome after DCH of the non-speech dominant hemispheric infarction includes significant cognitive deficits along with a physical handicap and impaired mood, and consequently, a diminished quality of life. The prospect of living with depression combined with severe physical and cognitive handicaps may be inacceptable for many patients; for others, however, it might be the better option compared to 70-80% mortality with a conservative treatment .not have opted for DCH did not differ significantly in their neuropsychological test results, and also did not display lower quality of life scores as compared to the patients who stated that they would again opt for DCH. However, the former patient group suffered from significantly higher scores for depressive mood (p = 0.039) than the latter. More than half of our patients were treated with anti-depressive drugs. It could be hypothesized that the patients whose post-hoc assessment of DCH was negative just needed a better anti-depressive treatment, especially since the agreement rates in our patient group were considerably lower than in other trials . Our daThe fact that in the majority of patients a significant cognitive decline is to be expected should not lead to a general rejection of DCH as a treatment option. The problem for the treating physician is to ascertain whether the patient is willing to risk death with conservative treatment or survive to a life of coping with impaired neurocognitive functions, probable depression and diminished quality of life. Ideally, this decision should be made by the patients themselves, which is too often not possible.The authors declare that they have neither competing financial nor non-financial interests with respect to this manuscript.HS conceived and coordinated the study and drafted the manuscript, TH carried out the neuropsychological examinations and took part in the study design, JE coordinated and carried out the statistical analysis, MD was involved in the study design and the neurological examinations, SH participated in the coordination of the study and drafted the manuscript, KN carried out the statistical analysis and helped coordinate the study, HP and AK participated in study design, and VR participated in the study design as well as coordination.All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/11/77/prepub


Many human diseases and phenotypes are related to RNA expression, levels of which are influenced by a wide spectrum of genetic and exposure-related factors. In a large genome-wide study of muscle tissue expression, we found that some genes exhibited a bimodal distribution of RNA expression, in contrast to what is usually assumed in studies of a single healthy tissue. As bimodality has classically been considered a hallmark of genetic control, we assessed the genome-wide prevalence, cause, and association of this phenomenon with diabetes-related phenotypes in skeletal muscle tissue from 225 healthy Pima Indians using exon array expression chips.GSTM1, HLA-DRB1, ERAP2, HLA-DRB5, MAOA, ACTN3, NR4A2, and THNSL2) were found to have bimodal expression replicated in the separate batch groups, while 24 other genes had evidence of bimodality in only one group. Some bimodally expressed genes had modest associations with pre-diabetic phenotypes, of note ACTN3 with insulin resistance. Most of the other bimodal genes have been reported to be involved with various other diseases and characteristics. Association of expression with cis genetic variation in a subset of 149 individuals found all but one of the confirmed bimodal genes and nearly half of all potential ones to be highly significant expression quantitative trait loci (eQTL). The rare prevalence of these bimodally expressed genes found after controlling for batch effects was much lower than the prevalence reported in other studies. Additional validation in data from separate muscle expression studies confirmed the low prevalence of bimodality we observed.Two independent batches of microarrays were used for bimodal assessment and comparison. Of the 17,881 genes analyzed, eight , and is much lower than limited reports from other studies. The major cause of these clearly bimodal expression patterns in homogeneous tissue appears to be  RNA expression levels indicate the overall effect of a combination of genetic and environmental factors ,2, whichOur current study measured RNA expression in the largest genome-wide analysis of human skeletal muscle tissue of which we are aware -17. In pfuture disease development. Previous reports of bimodality [Limited reports of bimodal expression patterns in other studies have also been made -20, partmodality ,21-23,27modality . As partWhile this discrepancy in the estimated prevalence of bimodality is large and invites further investigation, the ability to distinguish genuine bimodals can lead to the more accurate evaluation of their biological role. Bimodality is an important biological phenomenon as it implies the existence of discrete populations  or discrete genetic or environmental influences on a trait. As such, bimodality has historically been sought in genetic research, yet few reproducible bimodal patterns have ever been observed in this field. One might, therefore, hope that genuine bimodally expressed genes will have significant genetic importance. Hence, it is most intriguing that many of the bimodal genes we identify herein have reported associations with a variety of human diseases and phenotypes. This elicits a multitude of hypotheses regarding the role of bimodal expression and human characteristics, including disease development. Our analysis also evaluates the role of bimodality in one such disease - type 2 diabetes, by assessing the association of the identified bimodal transcripts with pre-diabetic phenotypes in this population of non-diabetic Pima Indians who are at high risk for developing diabetes in their lifetime.The prevalence of bimodal expression was estimated from skeletal muscle biopsies taken from 225 non-diabetic, healthy Pima Indians. These tissue samples were scanned on Affymetrix Human Exon 1.0 ST microarray chips which provide on average 49 expression level measurements per gene. Subject characteristics are shown in Table GSTM1, HLA-DRB1, ERAP2, HLA-DRB5, MAOA, ACTN3, NR4A2, and THNSL2) were found to meet the stringent bimodal criterion in group A, group B, and the total study of 225 samples. Figure 2 expression level distributions for each of these confirmed bimodal genes. An additional 10 genes identified as bimodal had significant association with gender, of which 9 were located on the Y chromosome  appeared to have RNA expression at two distinct abundance levels, each with lower modes corresponding to levels near or above the median of the high mode of ERAP2 (55th percentile).As bimodal expression may reflect either two distinct levels of transcript abundance or a single transcript abundance level with the lower mode reflecting no expression, we compared the median levels of the lower mode of the confirmed bimodal genes to the median expression levels of all other genes . The levels of the lower mode were indicative of background (no gene expression) for half of these confirmed bimodals - having median expression levels at the 25HLA-C, SLC44A5, NR4A3, HSPC157, ABCC6, and C19orf62) to have a false discovery rate (FDR) < 0.05. The p-values for bimodality of all 17,881 genes in both independent batch groups are plotted in Figure Many genes initially classified as bimodal in group A had very few data points (often 1 or 2) lying to the left or right of the intersection of the two component normal distributions. As discussed in Methods, genuine bimodality should not reflect the potential influence of a small number of outliers. For a robust reassessment of bimodality, we deleted the lowest 5% and highest 5% of expression values by chip group for each gene. Bimodality was again assessed in these trimmed data sets (n = 65 and n = 43), with similar bimodal prevalence estimates found to the estimate which required a minimum of 10% of data points to be in each component  and percent body fat to be higher in those in the high mode of MAOA expression (p = 0.018). Body mass index and acute insulin response to an intravenous glucose tolerance test were not found to be associated with any of the bimodal genes in this analysis.We assessed the association of the mode of expression with pre-diabetic traits , in the eight confirmed bimodal genes Table . We founCis-acting SNPs (defined as lying within a 200 kb region to either side of the gene location) were sought in this population. Nearly all of the confirmed bimodally expressed genes and half of the entire bimodal set had highly significant (unadjusted p < 10-8) associations between muscle tissue expression and SNP genotype . We further investigated this gene for any trans-determinant cause across the entire genome, but no SNP approached a genome wide significance level. We also noted that while GSTM1 had the clearest separation of expression modes, it had the lowest kappa coefficient of the seven cis-associated bimodals, possibly indicating influences other than nearby SNPs on the expression levels, such as environmental exposure or more complex genetic models.To investigate the potential cause of such bimodal expression, a subset of 149 individuals who were also genotyped (Affymetrix Genome-wide Human SNP Array 6.0) were analyzed for association with muscle expression levels. pe Table , and werpe Table . Still, GSTM1), for the majority of confirmed bimodals, known CNV is likely not the explanation for such a distribution.As polymorphic deletions may greatly affect expression levels, we sought to determine the frequency with which such copy number variation (CNV) may be responsible for producing the bimodal expression patterns. The genes listed in Table -8 of chance concordance of the bimodal gene sets in each of these populations with the Pima bimodals  did not show strict bimodality in these other populations, related genes were highly bimodal in these other data sets .We further assessed bimodal RNA expression in two publically available data sets containing expression data from healthy muscle biopsies , though still more frequent than occurs genome-wide. Hence in healthy individuals, eQTL are prime (yet not exclusive) locations for finding bimodally expressed genes. We also found that known copy number variation - while a potential explanation for some bimodal expression, was not found in higher abundance than across the genome, and is hence likely not the main source of such distributions.Our detection of bimodal gene expression in skeletal muscle tissue reveals an apparently rare (<0.2%), yet potentially new predictor of human disease and phenotypes. Of the eight confirmed bimodal genes, we found that seven were expression quantitative trait loci, as they were strongly to completely associated with possibly causal SNPs occurring within a 200-kb ACTN3 expression, with 42.2% of the individuals falling in the higher expressed mode which was associated with insulin resistance. While not meeting strict correction for multiple testing, this gene has previously been associated with slow- and fast-twitch muscle fiber, with correlation to athletic performance [ACTN3 could reflect a bimodal distribution in the proportion of type 2 fibers present in the subjects, though we are not able to assess this directly. As insulin resistance is a known predictor of diabetes [ACTN3 expression deserves further investigation.The current study also assessed the relationship of RNA expression mode with known predictors of diabetes. We observed a likely association of insulin sensitivity with mode of formance . The bimdiabetes , the rolHLA loci, as the HLA system is known not only to convey risk for a variety of immune-related diseases, but contains the main genetic risk factors for type 1 diabetes [HLA genes  were found to have bimodal distributions - as well as ERAP2 which plays a final role in producing HLA peptides [Many of the other bimodal genes identified have previously been reported to be associated with other diseases and characteristics ranging from cancer to arthritis -39, somediabetes . That thpeptides , is of gThe prevalence of bimodal gene expression we have observed in a healthy single tissue is approximately 100-times lower than what has been reported in mixed-tissue studies ,22. SincOur estimate also differs greatly from the only other prevalence reported for bimodal expression in a population comprised solely of healthy persons - 28% in lymphoblastoid cells ,28. As tWe highlight two technical issues for future studies to be aware of: use of a model that allows for unequal variances in the bimodal distribution and the determining of batch effects. We observed that a model that allows for unequal variances was clearly more appropriate than a constant variance model for approximately 20% of the genuine bimodal genes identified in our data, and hence the failure to allow for such as well as differences in data scaling may contribute to some variability in prevalence estimates. Even more important is the potential of batch effects to cause artifactual bimodal expression. Such confounding can occur when any set of experimental factors is not uniform - causing the expression levels to be uniformly over- or under-estimated for a given transcript for a portion of the samples. We noted that these batch effects were associated more frequently with genes of higher transcript abundance levels. We also identified that chips which had distinct lot number differences and which had been processed on distinctly different fluidics station sets gave rise to such artifactual bimodality, though it was not possible to determine which of these factors had a more primary role in this study. However, because this grouped source of heterogeneity was identified for most arrays, bimodality could be analyzed in homogeneous groups, eliminating not only the unwanted bias, but also providing distinct replication sets in which tests for bimodality could be confirmed.cis-polymorphisms, being expression quantitative trait loci. Future investigations of bimodality in homogeneous tissues from healthy persons will be of great interest in assessing similarities and differences across other tissues and populations, as well as their association with other human characteristics and diseases. In the Pimas, the association of a bimodal gene with insulin resistance and the presence of bimodality in genes from the HLA locus which carries the greatest risk for type 1 diabetes underscore the need to further investigate and understand this interesting phenomenon.In conclusion, the prevalence of bimodal RNA expression in homogeneous tissue is quite low, and hence has been unrecognized in most expression analyses. The few prior genome-wide studies which have reported bimodality may have over-estimated its prevalence, possibly due to the presence of batch effects and/or overly lenient statistical thresholds. Differences in cell type, microarrays, and populations may also be responsible for some of the large difference in estimated prevalence. An additional novel insight from this analysis is that the majority of genes with bimodal RNA expression apparently exhibited this pattern due to All subjects were part of our study of the etiology of type 2 diabetes among Pima Indians living in Arizona. Volunteers providing muscle biopsies were non-diabetic, healthy, and were not taking any medications. Subjects were assessed for diabetes-free status via a fasting blood draw as well as a 75 gram 2 hour oral glucose tolerance test (OGTT) to confirm that the subject was indeed non-diabetic (fasting glucose <126 mg/dl and 2 hour glucose <200 mg/dl). All subjects provided informed consent (which places some restrictions on access to data and intended research purposes) and all studies were approved by both the institutional review board of the National Institute of Diabetes and Digestive and Kidney Diseases and the council of the Gila River Indian Community.Most muscle biopsies were performed within one week's time of the diabetes assessment . After a 12 hour overnight fast and monitored physical inactivity, percutaneous needle biopsies were carried out on the vastus lateralis muscle under local anesthesia with 1% lidocaine. The biopsy specimens were snap frozen and stored at -20 to -70°C. Variation in storage time ranged up to 16 years, however no effect of the amount of time frozen on RNA quality was found.Muscle tissue samples were homogenized using a PT 1300D  homogenizer with 12 mm rotor/stator head in 2.0 ml of cold TRIzol reagent . The samples were then heated to 65°C and 0.4 ml chloroform  was added. 5 μL of 4 ng/μL Carrier RNA was added to each sample to aid precipitation with ETOH and further purified using RNeasy Micro Kit . 1 μg Total RNA Labeling Protocol starts with a ribosomal RNA (rRNA) reduction procedure where the 28S and 18S rRNA portion is significantly reduced from the total RNA sample minimizing the background and increasing the array detection sensitivity and specificity. All RNA was isolated by a single technician.The rRNA was prepared and the cDNA synthesized for the Human Exon 1.0 ST Array microarray chips  using the GeneChip Whole Transcript Sense Target Labeling Assay kits (Affymetrix). Chip washing and staining was performed in two distinct sets of fluidics stations  and scanned on as many as five different scanners . Two technicians performed the scanning measurements in discrete sets of dates.Quality control metrics and technician insight were used to determine the need for re-scans. When re-scanning did not resolve the problem, such chips were discarded and cDNA hybridization mix placed on completely new chips (this occurred in 9.8% of the samples). A total of 225 baseline samples which passed such quality control metrics resulted in expression results available for analysis. Only transcript clusters identified as "core" (genes which have been identified with a high level of confidence) were analyzed, comprising a total of 17,881 genes.2 intensities with prior GC correction , usi, usi2 inIn analyzing the 225 muscle files, we discovered a batch effect which was associated with distinctly different expression values by clusters of sample processing dates for some genes . While differences between clusters were observed for some genes, other genes appeared to be free from this batch effect. For these unaffected genes, power to detect associations with true bimodality would be greater in the total data set (225 chips) due to increased sample size. Hence, we first confirmed bimodality in the separate clusters before using the entire data set for further confirmation and association analyses.2 expression data for each gene were transformed by the Box-Cox method to reduce skewness [1, σ1, μ2, σ2) and the proportion of area in one of the components . Such methods for model fitting have been used previously [For each gene, we had 47, 71, or 225 data points corresponding to the expression levels coming from each participant in the two batch groups or in the entire study. To avoid skewness with a unimodal distribution resulting in an artifactual inference of bimodality, the logskewness . The Boxeviously ,44. A poThere is no unequivocal selection test for bimodal versus unimodal distributions, despite the fact that the models are nested ,24,45. HAdditionally, we introduced a new requirement that the minimum number of data points to the left and right of the selected intersection point be at least 10%. This prevented bimodal model selection when a single or small number of outliers would otherwise have caused bimodal selection. Such a requirement is necessary to eliminate potentially large numbers of bimodal misclassifications for genes in which the best fit distribution's misclassification area is not significant, yet the bimodal fit is clearly the result of a single or small number of outlier points. As a consequence, this additional criterion will permit only the detection of bimodal genes that are dichotomously expressed in a non-trivial percentage of individuals, yet with greater confidence that these genes are not mis-assessed due simply to outlier points.We confirmed the need for such a strategy in a separate analysis in which the lowest 5% and highest 5% of expression values were dropped. Such a strategy would prevent misclassification of bimodality due to a small number of outliers, though it would reduce the power to detect such in clean data. We noted this method to give very similar end-result prevalence estimates.As some genes are expressed differently by gender, we fit a separate linear model on GC-RMA normalized expression levels to assess this association, using the resulting two-sided p-value as an additional tool to determine which genes exhibit bimodal expression due to different expression levels in men and women. If this p-value was ≥ 0.05, it was assumed that the bimodality was not due to gender.cis-acting SNPs  were idAssessment of pre-diabetic traits was made as part of the research protocol corresponding to the investigation nearest to the muscle biopsy, usually performed on the same day, and never greater than 30 days from the biopsy date. Insulin action in vivo was assessed using the hyperinsulinemic, euglycemic clamp as described in . Percenthttp://www.ncbi.nlm.nih.gov/geo/). We re-analyzed bimodality in the expression of lymphoblastoid cells from 193 CEPH Utah residents  and 85, ,28 aGSE1485, ). The exGSE1485, ,28. ThisGSE1485, .Fisher's method was used to combine p-values from independent chip groups. False Discovery Rates  were assGene scans were performed using Affymetrix GCOS software and Affymetrix Expression Console. GC-RMA normalization was performed using Partek Software Version 6.4 (6.09.0422). SAS Version 9.1 was used for Box-Cox transformations, Newton-Raphson maximum likelihood convergence, pre-diabetic phenotype associations, Fisher's exact test, as well as for all SNP association analyses. Code was also written in Microsoft Visual Studios C++ for data manipulation and maximum likelihood with automatic parameter estimation distribution fitting of unimodal and bimodal models.Data were acquired by CCM, CB, JK, LB, and VO; data were analyzed by CCM; interpretation of the data was made by CCM, RLH, and CB; CB and LJB provided funding; CCM conceived the bimodal study, designed the figures, and wrote the paper, with advice given from CB and RLH. All authors read and contributed to the manuscript.Figure illustrating how dichotomous gene level RNA expression can be artifact of batch effect.Click here for fileSummary of 16 genes (associated with gender) which met all criteria for bimodality in either group A or group B.Click here for fileSummary of bimodal genes found on trimmed bimodal data sets of non-gender associated genes. Listed are 28 genes which had a false discovery rate on the Fisher combined p-value from the two trimmed data sets <1.0. Fourteen of these have an FDR < 0.05.Click here for fileSummary of bimodal genes found on trimmed bimodal data sets of gender associated (p < 0.05) genes. Listed are 30 genes which had a false discovery rate on the Fisher combined p-value from the two trimmed data sets <1.0. Twelve of these have an FDR < 0.05.Click here for file-8 in the 32 bimodally expressed genesList of 211 SNPs associated with component of muscle expression with p < 7 × 10.Click here for fileComparison of bimodal gene sets found in the various populations analyzed.Click here for file