The distribution of PIP2 has been analyzed extensively using live imaging of the GFP-coupled phospholipase C-δ1 pleckstrin homology domain in cultured cell lines. However, technical difficulties have prevented the study of PIP2 in cells of in vivo tissues. We recently developed a method to analyze the nanoscale distribution of PIP2 in cultured cells by using the quick-freezing and freeze-fracture replica labeling method. In principle, this method can be applied to any cell because it does not require the expression of artificial probes. In the present study, we modified the method to study cells of in vivo tissues and applied it to pancreatic exocrine acinar cells of the rat. We found that PIP2 in the plasma membrane is distributed in an equivalent density in the apical and basolateral domains, but exists in a significantly higher concentration in the gap junction. The intracellular organelles did not show labeling for PIP2. The results are novel or different from the reported distribution patterns in cell lines and highlight the importance of studying cells differentiated in vivo.Phosphatidylinositol 4,5-bisphosphate [PIP PIP2 is also the precursor of inositol-1,4,5-trisphosphate, phosphatidylinositol 3,4,5-trisphosphate [PIP3], and diacylglycerol, which all play critical roles in signal transduction 2 exerts its multiple functions, we must define the distribution of PIP2 on the smallest possible scale using quantitative methods.Phosphatidylinositol 4,5-bisphosphate [PIP22 that is bound to effector proteins, and the influence of inositol-1,4,5-trisphosphate, which has a higher affinity for GFP-PH than PIP2. Furthermore, the GFP-PH imaging method cannot be readily applied to cells in vivo because efficient transfection of GFP-PH cDNA is not easy.Live imaging using the GFP-tagged pleckstrin homology (PH) domain of phospholipase C (PLC)-δ1 as a probe has greatly advanced our understanding of the functionality of PIP2 may be examined using conventional immunolabeling techniques 2 that may occur during the labeling procedure is a potential problem, especially for accurately examining PIP2 localization at the small scale. This is mainly because aldehyde fixatives that are used for sample preparation do not react with most membrane lipids, and therefore, membrane fluidity persists even after fixation Alternatively, the distribution of PIPTo circumvent the fixation problem, we recently developed a physical method to fix membrane lipids using quick freezing and freeze-fracture replica formation 2 in rat pancreatic exocrine acinar cells in the current study. These cells form a simple epithelium with distinct apico-basolateral polarity and have well-developed intercellular junctions. With regards to the implication that PIP2 is related to cell polarity formation 2 distributes in the epithelium differentiated in a physiological environment. We modified the original QF-FRL method in several respects to analyze cells taken from in vivo tissues. By using the modified QF-FRL method, we examined the detailed PIP2 distribution in the rat pancreatic acinar cell, and obtained several novel results as well as results different from those previously reported in cell lines in culture.By taking advantage of the versatility of the QF-FRL method, we examined the distribution of PIPAll animal treatments in this study conformed to the Guidelines for Proper Conduct of Animal Experiments of Science Council of Japan and this study was approved by the Animal Experimentation Committee of Nagoya University Graduate School of Medicine .2E. coli and affinity-purified using a glutathione-agarose column as previously described Mouse PLC-δ1 was used as a template to prepare recombinant GST-PH protein and its mutant version GST-PH, which does not bind to PIP3) in Tyrode's solution, placed on an aluminum disc (3 mm in diameter) with a shallow indentation (0.3 mm depth), and covered with a thin stainless steel foil (10 µm in thickness). The sample assembly was frozen using an HPM 010 high-pressure freezing machine (Leica) according to the manufacturer's instructions. The procedure from excision to freezing generally took at least several minutes. For freeze-fracture, the specimens were transferred to a cold stage of a Balzers BAF 400 apparatus and fractured at −130°C under a vacuum of ∼1×10−6 mbar. Replicas were made by electron-beam evaporation of carbon (C) (∼5 nm in thickness), followed by platinum (Pt) (2 nm) and then by C (20 nm) as described before Pancreas was excised from adult male Wistar rats under anesthesia. The animals were fed ad libitum and thus the upper gastrointestinal tract contained digested food. Pancreas was minced with a razor blade to small pieces  at 60–70°C overnight. Following the treatment described above, replicas were stored in buffered 50% glycerol at −20°C until use.Thawed replicas were treated with 2.5% SDS in 0.1 M Tris-HCl (pH 7.4) at 60–70°C for 30–60 min and digested with 0.25% trypsin and 0.5 mM EDTA in PBS for 1 h at 37°C. After washes with 1% Triton X-100 in PBS (PBST), the samples were treated with 100 µg/ml of DNaseI (Sigma) in a solution containing 50 mM Tris-HCl (pH 7.5), 1 mM MgClFreeze-fracture replicas were washed with PBST, blocked with a mixture of 3% BSA and 2% cold fish skin gelatin in PBS, and incubated with GST-PH (67 ng/ml) in 1% BSA and 1% cold fish skin gelatin in PBS at 4°C overnight. The samples were subsequently treated with rabbit anti-GST antibody (10 µg/ml) followed by colloidal gold (10 nm)-conjugated protein A (1∶60 dilution of the supplied solution), both for 30 min at 37°C in 1% BSA in PBS. After each incubation, replicas were washed extensively with 0.1% BSA in PBS. After brief rinses with distilled water, replicas were picked up on formvar-coated EM grids and observed with a JEM-1011 electron microscope operated at 100 kV. Digital images were captured by a CCD camera (Gatan Inc.) and subjected to further analysis. The binding specificity of the GST-PH probe in the labeling method was verified previously by using freeze-fracture replicas of liposomes containing different phosphoinositides t-test.Electron micrographs obtained from more than three independent experiments were used for the analysis. The areas of interest were measured using ImageJ (NIH), and the number of colloidal gold particles was counted manually. Statistical differences between samples were tested using the Student's 2 and gangliosides GM1 and GM3 in vivo have several critical differences from cells in culture, we needed to modify the method in several respects Prespects .First, the cells cultured in a monolayer are usually less than 10–20 µm in vertical thickness, whereas tissue pieces are much thicker. Therefore, to freeze the samples without ice crystallization, we substituted the metal contact method for the high-pressure freezing method Second, the volume of cell and tissue fragments that adhere to the freeze-fracture replica is far greater in tissue pieces than that in cultured cells. The treatment in the SDS solution is not sufficient to remove all of the extramembrane materials from the replica of tissue specimens, which is problematic because the remnants tend to induce the artificial binding of probes. To efficiently remove extraneous materials, the replicas were cleaned using trypsin and DNase I before the SDS treatment. The trypsin and DNase I digestion did not affect the labeling of the membrane lipids, which was evaluated by comparing replicas of cultured cells with and without this treatment (data not shown).in vivo tissue samples.Third, a mixture of cold fish gelatin and BSA rather than BSA alone was used to block the samples and dilute the GST-PH probe. The combination of the two proteins was effective in reducing non-specific labeling in replicas of 2 in the pancreatic acinar cell was labeled with sufficient specificity and intensity. The distribution density of colloidal gold particles that label PIP2 in the basolateral plasma membrane of the epithelial cell was 303/µm2 when GST-PH was used at the concentration of 67 ng/ml, which was less than 422/µm2 in cultured human fibroblasts at the GST-PH concentration of 30 ng/ml 2 concentration in the two cell types. We have observed that different types of cultured cells show highly variable labeling densities .With these modifications, PIPin vivo properties are maintained: 1) Pancreatic acini obtained by dissection followed by extensive enzymatic treatments exhibits various physiological reactions 2, a similar caveolar concentration was observed in smooth muscle dissected from mouse vas deference and in cultured human fibroblasts At least several minutes were needed to dissect the pancreatic tissue from anesthetized rats and freeze it, but the following literature suggests that most 2, whereas the E face was scarcely labeled  and outer  leaflets of the membrane are represented as the P and E faces, respectively. The two faces are distinguished based on differences in the distribution density of intramembrane particles  and the ultrastructure of the surrounding areas . In the pancreatic epithelium, the P face of the plasma membrane was densely labeled for PIP labeled . The lababeled 2.The pancreatic acinar cells comprise the highly polarized simple epithelium. The apical and basolateral domains of these cells are clearly demarcated by the well-developed tight junctions. The two membrane domains were distinguished unambiguously based on the tight junction and the apical microvillus in freeze-fracture electron microscopy. The tight junction in the quick-frozen specimens was observed as intermittent particles in the E face and as a shallow groove in the P face , Fig. S12 in the P face did not show differences between the apical and the basolateral domains P2 in the apical membrane distributed without distinct local concentration. The membrane in the tight junction area was also labeled in a similar density as the apical and basolateral membranes P domains . In the  domains , but somembranes .2 in the lipids of the inner leaflet is quite high in the gap junction.Gap junctions, which were identified unambiguously as plaques of densely-packed intramembrane particles in the P face, were labeled more densely than the surrounding basolateral membrane , Fig. S2The pancreatic acinar cells are also highly polarized in the disposition of intracellular organelles. Multiple layers of the rough endoplasmic reticulum (ER) and a cluster of large secretory granules are present in the basal and apical cytoplasm, respectively. This polarized disposition and characteristic morphology, which reflects the cellular exocrine activity, was used to identify the organelles in freeze-fracture replicas.2 was not observed in any organelle in the P and E faces P E faces . Organelmembrane , the ER membrane , the Golmembrane , the secmembrane , and themembrane . In the membrane . Therefo2 in cultured cells have been studied extensively using live imaging with PH domains that have been tagged with GFP 2 was observed to be enriched in the apical membrane 3 was confined to the basolateral membrane 2 and PIP3 were implicated as the apical and the basolateral membrane organizers, respectively Intracellular distribution and dynamic movement of PIP2 in the pancreatic acinar cells in the adult rat was detected in equivalent densities in the apical and basolateral membranes. This result may initially appear to contradict the findings in MDCK cells, but the difference may be explained in several ways. First, the MDCK model probably mimics a developing stage when the apical lumen is in the process of being formed. Therefore the patterns that are evident in the MDCK model may differ from those observed in the established epithelium in the adult rat tissue. Second, the pancreatic acinus and the MDCK cyst may use different mechanisms for apical lumen formation. Although the cord hollowing process in MDCK cells 2 population that is not bound to effector proteins, and may not reflect the density of total PIP2. This is likely to happen because whether and to what extent GFP-PH replaces endogenous PIP2-binding proteins has not been known. Therefore the PIP2 density in the apical and basolateral membranes of MDCK cells may not be so divergent as suggested by live imaging.In the present study, PIP3 to produce PIP2, was previously identified as a critical event in the formation of the apical domain 2 is as much as 1,000 times higher than that of PIP32 that is generated by PIP3 hydrolysis constitutes only a very minor proportion of the total PIP2 and should not have much significance 2 that is generated at the right location at the right time is more important in the cell polarization process than the total amount of PIP2. To test this conjecture and elucidate the true PIP2 distribution, it would be helpful to study the same cell system using both GFP-PH imaging and QF-FRL.All of the above possibilities are reasonable and are not mutually exclusive, but we think that the third possibility is the most plausible explanation of the results. Cdc42 is the master regulator of cell polarity 2 was more intense in the gap junction plaque than in the surrounding membrane. Because the lipid-protein ratio is lower in the gap junctional membrane than in the general membrane 2 in phospholipids of the gap junction must be even higher than that indicated by the labeling density. This concentration of PIP2 in the gap junction is intriguing, because cell-cell communication through the connexin 43 (Cx43)-based gap junction channel is regulated by the local concentration of PIP22 upon receptor activation The labeling for PIP2 to regulate the gap junction is not known, but its direct binding to connexin molecules is a possible mechanism. Cx43 may bind to PIP2 through a putative PIP2-binding domain in the C-terminal domain, which contains positive charges that are interspersed with hydrophobic residues 2-binding domain and may bind PIP2 directly.The mechanism used by PIP2 in the gap junction suggests that the channel is in the open state in the pancreatic acinar cell observed in the present study. It would be interesting to study whether the density of PIP2 changes when challenged with various reagents. The present method would be a useful tool for further analyses.The abundance of PIP2 in the nuclear membrane, the ER membrane, and the mitochondrion was expected. However, the absence of PIP2 labeling in the Golgi membrane was puzzling because the function of PIP2 in membrane trafficking around the Golgi has been previously demonstrated 2 in the Golgi was observed in ultrathin cryosections 2We observed more than 300 cross-fractured cell profiles, but no positive labeling was detected in any organelle. The absence of PIP2 in the liposomal membrane can be detected 2 may have hampered its detection with the QF-FRL method. Because the plane of freeze-fracture seldom runs along membranes that are bent acutely, indentations or protuberances are not well represented in many samples. We speculate that if PIP2 exists in the Golgi, it does not distribute evenly but is concentrated in a specialized compartment.The sensitivity of the current method should not be the cause of the negative results because as low as 0.5 mol% of PIPIt is unclear why non-specific binding of GST-PH occurred in the secretory granule membrane. The PH domain of PLCδ1 has been used in many experiments, but non-specific binding has not been reported, as far as we know. However, most experiments have been done using isolated lipids and the possibility that the PH domain binds to certain non-lipid molecules is difficult to refute. This reinforces the importance of always running a control experiment using the GST-PH mutant.in vivo tissues to observe the distribution of PIP2. The results showed that PIP2 in the pancreatic acinar cells was evenly distributed in the apical and the basolateral membranes and that PIP2 was locally concentrated in the gap junction. These results may be difficult to obtain using other methods available at present. It is always important to study cells that differentiate in a physiological environment. By expanding the applicability to other membrane lipids, the QF-FRL method may become an even more useful tool for such analyses.The present study demonstrated that the modified QF-FRL method can be successfully applied to cells taken from Figure S1The tight junction. The tight junction was observed as strands in the E face that separate the apical (Ap) and the basolateral (BL) membrane domains. The labeling for PIP2 was observed in the P face of the plasma membrane. The apical domain is only observed in the E face.(TIF)Click here for additional data file.Figure S2The gap junction. The same micrograph as (TIF)Click here for additional data file.


Acyl chain remodeling in lipids is a critical biochemical process that plays a central role in disease. However, remodeling remains poorly understood, despite massive increases in lipidomic data. In this work, we determine the dynamic network of ethanolamine glycerophospholipid (PE) remodeling, using data from pulse-chase experiments and a novel bioinformatic network inference approach. The model uses a set of ordinary differential equations based on the assumptions that (1) sn1 and sn2 acyl positions are independently remodeled; (2) remodeling reaction rates are constant over time; and (3) acyl donor concentrations are constant. We use a novel fast and accurate two-step algorithm to automatically infer model parameters and their values. This is the first such method applicable to dynamic phospholipid lipidomic data. Our inference procedure closely fits experimental measurements and shows strong cross-validation across six independent experiments with distinct deuterium-labeled PE precursors, demonstrating the validity of our assumptions. In constrast, fits of randomized data or fits using random model parameters are worse. A key outcome is that we are able to robustly distinguish deacylation and reacylation kinetics of individual acyl chain types at the sn1 and sn2 positions, explaining the established prevalence of saturated and unsaturated chains in the respective positions. The present study thus demonstrates that dynamic acyl chain remodeling processes can be reliably determined from dynamic lipidomic data. Lipids are fundamental building blocks of cellular membranes and are also essential for signal transduction, energy homeostasis, and many other cellular processes. Recent advances in mass-spectrometry have made large-scale quantification of lipidomes possible Glycerophospholipids are the dominant lipids in mammalian membranes and are comprised of a glycerol moiety, a polar head group linked via a phosphate to the sn3 position of the glycerol moiety as well as an acyl chain esterified to the sn1 and the sn2 positions Traditionally, the acyl remodeling process has been studied by addressing the specificity of individual enzymes in vitro using a limited number of substrate species In particular, pulse-chase experiments with isotope-labeled precursors (such as choline or ethanolamine) should provide superior information to steadystate measurements To more effectively analyze this type of data, we have now constructed a novel predictive method for determining the lipid remodeling network and its parameter values. We demonstrate the usefulness of this approach by analyzing data from pulse-chase experiments with 6 different exogenous phosphatidylethanolamine precursors, i.e. 14:0-14:0, 14:1-14:1, 18:3-18:3, 18:0-18:1, 18:1-18:1, 18:0-22:6 Our model assumes that (1) sn1 and sn2 acyl positions are independently remodeled, (2) remodeling reaction rates do not vary over time and (3) the concentrations of the acyl donors are constant. These assumptions, which are based on previous findings on acyl remodeling of phosphatidylethanolamine, phosphatidylcholine and cardiolipin We analyzed a set of 6 separate pulse-chase experiments carried out previously see . In thosWe consider the PE remodeling system as a chemical reaction network, defined by a finite directed graph Remodeling in cells is likely to proceed primarily through a subset of the possible edges in the full network. The first step toward the identification of the dominant remodeling pathways was taken in ref. evel See .Once a correlation network has been inferred for a given experimental timecourse, the behavior of the system can be forward simulated using ordinary differential equations (ODEs) determined by the reduced bidirectional correlation network. ??ions See . As can ions See . More geIn addition, we observed that fits were systematically worse when we used parameters other than those that were found in the correlation network inference step. We observed that when we removed the parameters corresponding to the major reactions  are consistent , and 1 : 0.26 : 0.24, respectively. Thus these ratios are relatively robust.  This behavior indicates that common mechanisms are active in separate precursor experiments. Therefore, cross-validation using independent precursors should be generally effective for determining model robustness. The observed consistency of rates also indicates the accuracy of our methodology.More generally, we were able to distinguish deacylation and reacylation rates and determine relative rates for different chain types by integrative analysis of the combined data using the steady state approximation for the concentrations of reaction intermediates See . For exaRobust deacylation and reacylation rates were also found for the sn2 position, although there were less data than for the sn1 position. For example, the relative conversion rate of 14:0 to 18:1 vs. 18:2 was  :  See . This orWe also observed consistencies in secondary effect parameters. For instance, the sn1 16:1 to 18:1 conversion rate was found to be Due to the complexity of lipid remodeling and the lack of previous computational tools, quantitative interpretations of lipidomic data have been rare By applying our new method, we have revealed a number of important facts about lipid remodeling. We have shown that 18:1 is the predominant reacylated acyl chain at both sn1 and sn2 positions, consistent with previous findings To appreciate the importance of the computational modeling approach we have developed, it is useful to compare the results with those that would have been deduced by empirical observation of the data. Empirical interpretation also has other shortcomings. For example, while the prior empirical interpretation suggested several interactions between sn1 and sn2 chains A strength of our method is that it is based on the straightforward position-independence assumption http://nbidiaz.github.com/DLipid/.In summary, we have constructed a powerful modeling tool for the analysis of glycerophospholipid remodeling pathways and their kinetics. The present work demonstrates that computational methods can quantitatively determine the details of glycerophospholipid remodeling by identifying the specificity and kinetics of deacylation and reacylation. Notably, the present method can be readily extended to other glycerophospholipid classes and should thus allow one to obtain a comprehensive picture of lipid remodeling, which will be essential for understanding lipid homeostasis in mammalian cells. A natural future application will be determining the mechanistic impact of various types of perturbations, e.g. knockdown of putative remodeling enzymes. Software implementations for the Correlation Network (JAVA) and Dynamic Simulation (MATLAB) are available at The remodeling pathway of phospholipids can be thought of as a directed weighted graph hth time point, respectively.Denote the set of candidate sources at the For each candidate source orithm 1 . We seleSingular Value Decomposition, SVD, or QR decomposition as described in The remodeling process of PE is a dynamical system that can be modeled by a set of coupled differential equations. This dynamical system depends on the rates of conversion of PE species into one another. These rates are not known a priori. Denote these parameters by the vector  process . Some pa process .As an additional verification, the inferred parameters were used to independently solve the differential equation using standard numerical techniques. In our implementation we used the matlab function ode45 with stepsize 0.01 with initial conditions and remodeling rates as solved by the dynamic algorithm.Deacylation and reacylation rates were calculated from the The relative ratios between rows and columns can be formally shown to indicate relative deacylation and reacylation rates by consideration of the kinetics of all species and deacylated intermediates using the steady state approximation for the intermediates. This approximation is justified by the fact that fully acylated PE is more prevalent than lyso- species in typical cells. Since the sn1 and sn2 positions are generally independent, we can consider the behavior of the sn1 (or sn2) position alone. For each chain type The kinetics of remodeling were derived from data obtained by using neutral loss scanning which allows selective detection of labeled vs. unlabeled PE molecules, but does not provide definitive information on the acyl substituents or their sn-positions. However, such information was obtained in separate experiments in which the cellular PE molecules were fragmented using collisionally-activated decomposition and the products were analyzed as described in Supporting Information S1Supporting information.(PDF)Click here for additional data file.


MSH2, MLH1 and MSH6 genes that belong to the DNA MisMatch Repair (MMR) genes family. The French network counting the 16 licensed laboratories involved in Lynch syndrome genetic testing developed three locus-specific databases with the UMD® software  that presently contain a total of 7047 sequence variations including 707 distinct variations of a priori unknown functional significance (VUS) that were identified through complete mutation screening or targeted predictive testing. Mutation carriers are at high risk for developing early-onset colorectal and endometrial adenocarcinomas. Consensus clinical guidelines have been proposed, allowing the efficient detection of curable lesions. The major challenge of genetic testing is to reliably classify the genomic variations in those patients who seek genetic counseling. Combining the interactive tools of the software, the relevant published data and mainly original information produced by the French MisMatch Repair network, the UMD-MLH1/MSH2/MSH6 databases provide interpretation data for the 707 VUS that were classified according to the IARC 5-Class system. These public databases are regularly updated to improve the classification of all registered VUS, exploring their role in cancer pre-disposition based on structural and functional approaches.Lynch syndrome is an autosomal dominant disease caused by germ line heterozygous mutations mainly involving the  MSH2, MLH1, MSH6 and PMS2) are implicated. To date, most of the causative mutations have been identified in two of them, MSH2 and MLH1. It has been estimated that the prevalence of mutations in either of these two genes in Western countries is between 1 of 500 and 1 of 1000. Monitoring of microsatellite DNA instability (MSI) in tumor cells provides good sensitivity but much lower specificity for the diagnosis of the syndrome. This instability is caused by the bi-allelic inactivation of the MLH1, MSH2, MSH6 or PMS2 genes in patients who may or may not carry a germ line mutation in these genes.Mismatch repair (MMR) genes mutation carriers are a subgroup of colorectal cancer prone individuals who, when identified, benefit from highly effective risk management . The genAmsterdam criteria have been first established to identify such condition based only on patients’ personal and family history of colorectal cancer. These initial criteria were too stringent to detect Lynch patients in medical practice and were progressively enlarged to include the MSI status of an extended spectrum of tumor types . Indeed,a priori classified of unknown functional significance (VUS) and require further investigation to document their impact on the MMR function. To refine the functional consequence of such variations, several criteria are generally used. In silico predictions first give an orientation toward a more likely neutral or causal effect. In theses studies, degree of conservation at the variation position among species and gene families, physico-chemical consequences on the predicted variant protein and splicing effect are investigated . Two curators collect and compile information from all 16 laboratories. The UMD-MLH1/MSH2/MSH6 databases centralize all identified variations whether causal, neutral or VUS; these variations are linked together through a unique sample ID, allowing the simultaneous retrieval of all genotypes reported in the databases for a given sample, called co-occurrences. In June 2012, the UMD-MLH1/MSH2/MSH6 databases contained data from 2389 entries for MLH1, 2380 for MSH2 and 1711 for MSH6. In this report, we first analyze variations observed in the French population. Then, we describe the 707 distinct MLH1, MSH2 and MSH6 VUS and the associated clinical and biological data. Finally, we explore the co-occurrence data in an attempt to classify VUS as either causal or neutral.Mutation databases can help in the classification of VUS by providing a compiled source of a large amount of information. We present here the databases of all genetic variations encountered by the French MMR network, which is made of the 16 licensed laboratories in France involved in the molecular characterization of Lynch syndrome, i.e. the MMR genes germ line analyses. These databases were developed with the universal mutation database UMD® software . They hahttp://www.umd.be/, UMD: PMID10612827 and PMID16086365)  . The UMDMLH1, MSH2 or MSH6 genes  and the Lynch syndrome. The ‘mutations’ and ‘statistics’ options allow the user to search into the databases. Hyperlinks are provided to access the data search options. The sixth part, ‘references’, aims at providing a list of all published references related to the VUS reported in the databases. It is currently in the validation process. The web pages also contain links to external information including other UMD databases and public central databases .Each website is divided in six parts. The ‘gene’, ‘protein’ and ‘clinics’ are dedicated to information on the The UMD knowledgebase system includes many analytical tools , 11. TheAccording to the French law #78-17, both project and on-line publishing were approved by the French supervisory authority CNIL  and the national ethics committee CCTIRS . About 20 000 index cases were addressed to the French medical genetics departments between January 1995 and June 2012 for investigation of possible Lynch syndrome. Genetic testing was performed according to the eligibility criteria stated by the French consensus . All patMLH1, MSH2 and MSH6 genes were screened for variations, based on prescreening methods or more recently by systematic exon sequencing. Large genomic rearrangements were identified by Multiplex Ligation-dependent Probe Amplification or Quantitative Multiplex PCR of Short Fluorescent Fragments. The corresponding references are displayed in the ‘clinics’ section. The French MMR network of laboratories involved in the genetic testing of Lynch syndrome collected all MMR variations identified in blood samples of the affected patients and at-risk relatives.The full coding sequences and exon–intron junctions of the MLH1, MSH2 and MSH6 genes were NM_000249.3, NM_000251.1 and NM_000179.2, respectively, as mentioned in the Locus Reference Genomic database. Genomic variations were described according to recommendations of the human genome variation society (www.hgvs.org/mutnomen/), and the predicted proteins were deduced using the algorithms of the UMD core system. Data are freely accessible at www.umd.be/MLH1/, www.umd.be/MSH2/ and www.umd.be/MSH6/. An anonymous and unique sample identifier (sample_ID) was generated for each individual and was used in the three databases. In June 2012 release, 2562 entries were recorded for MLH1, 2731 for MSH2 and 1754 for MSH6. The UMD software automatically checked for the correct description of the sequence variations at the nucleotide level and generated the variation name at the protein level. Collection of phenotypic data is in progress as well as pictures of Sanger-sequencing profiles. As the treatment of genomic events like large genomic deletions, insertions or deletions surrounding splicing junctions, deletion/insertions and variations located in the 5′UTR and 3′UTR regions, was recently integrated to UMD database structure, these variations are currently in the validation process and will be soon uploaded.Quality control procedures were in place throughout the collection, processing and storing of data as required by the standard ISO 15189 in all laboratories. Genotypes were double checked on two independent biological samples from the same individuals except in case of index case death before availability of genetic result. Reference sequences for the MLH1/MSH2/MSH6 databases are regularly reviewed. Information is individually available by clicking on ‘I found a mutation’ of the ‘mutations’ section. It will be soon fully accessible in the ‘references’ section.The system of five variation groups developed at the International Anticancer Research Center was used for functional classification . Mutatioin silico prediction, including splicing analyses, conservation level, SIFT and UMD-Predictor estimates were directly integrated into the UMD database structure. They are automatically activated when opening the summary of the corresponding variation.For each VUS, Cosegregation analyses were performed in the relevant families whenever possible, and the cumulative LOD scores were calculated, but the corresponding pedigrees were not displayed to keep data anonymous;Clinical phenotype was detailed for each patient according to the enlarged Amsterdam criteria;Assessment of the MMR function in tumor cells by genotyping and immunohistochemistry analysis. MSI/MicroSatellite Stability (MSS) classification followed the consensus recommendations and was validated only if the proportion of cancer cells in the tumor fragment exceeded 10%. Immunohistochemistry was performed using antibodies against the two proteins MSH2 and MLH1, completed with MSH6 and PMS2 when possible;et al.  was required;Splicing impairment was explored using a functional splicing assay developed and validated by Gaildrat et al. . In caseTranscription-translation assays were performed for VUS involving the Kozak sequence or the 5′UTR.In addition, five pieces of information were asked to French contributors then evaluated according to their influence on MMR function impairment :Cosegrein vitro or in/ex vivo assays according to the classification proposed by Ou et al.  in 171 (40%) occasions on MLH1, 199 (44%) on MSH2 and 97 (33%) on MSH6. A total of 707 variations thus remained VUS.In June 2012, the 16 licensed laboratories belonging to the French MMR network reported 2562 variations for Corresponding data can be sorted out as tables or individually viewed in the ‘mutations’ section. A graphical view of the mutations distribution along the coding sequence can be displayed in the ‘statistics’ section .Figure MLH1 gene, 256 on MSH2 and 193 on MSH6. In either the ‘mutations’ or ‘statistics’ option, in silico estimates can be displayed when clicking on a variation/variation type  in a table or when asking for a specific position in the coding sequence or when searching using the different modules. When the non-truncating variation occurs within the coding sequence, the UMD-Predictor tool gives first an estimate of the functional impairment based on structural alteration of the corresponding protein, pathogenicity at the physic-chemical level together with the conservation level, the SIFT prediction and a graphical view of the amino acid involved in the variation  is mentioned with the validation date of information. When available, data published by other groups are also displayed . It is reviewed when new data become available and only the most recent conclusion is presented and time stamped  and 3. TMLH1/MSH2/MSH6 variations collected by the 16 licensed laboratories located in France and belonging to the French MMR network during the past 18 years. Updates on new variations or new samples found to carry MMR variations are done twice a year. In June 2012, a total of 7047 variations were provided for registration, and 6480 could be integrated by the UMD software for further analyses. They represent 1174 different variations corresponding to 467 truncating mutations (40%) and 707 VUS (60%). Entries correspond to all variations found through a complete exon screening of the three genes not only in index cases but also in those relatives that were genetically screened and found to be mutation carriers. This nation-wide and systematic registration should improve the prevalence estimate of germline MMR mutations in France. We do not know yet to what extent the index cases carrying the same mutation are genetically related, as haplotypes carrying the variant allele have not been constructed. We plan to progressively get haplotypes for all index cases from the French providers, an information that could help in obtaining reliable demographic parameters, in estimating mutation rates and in improving allele-specific lodscores.We examined A total of 337 of 707 non-truncating French variations remain VUS (48%) even with information reported in other databases and published works . Additional experiments for all variations with uncertain functional status are requested from contributors. The most significant findings are used to change the functional status of the corresponding VUS according to the classification method previously proposed .www.insight-group.org). A dedicated database (www.mmruv.info) is linked to the previous one and contains the great majority of data available in the literature, but does not provide its own interpretation  database gives an exhaustive description of the data submitted by the contributors  neutral or causal. This classification has been achieved based on original data collection from patients referred to the French MMR network. The tool also displays all co-occurrences within the three genes on each VUS-specific page of the UMD-MLH1/MSH2/MSH6 databases. Information contained in the ‘mmruv’ database is available through a specific link on the same page. Pending data are progressively added, and attempt to classify the remaining 337 VUS is pursued. Therefore, the UMD-MLH1/MSH2/MSH6 databases consist in the most complete and comprehensive public tool for geneticists and molecular biologists to interpret their own sequencing results.The UMD-MLH1/MSH2/MSH6 databases presented here are an integrated system, allowing the simultaneous access to selected published data and original information provided by the French MMR network in the three


Golgi-localised, γ-ear containing, ADP ribosylation factor-binding) are a family of clathrin adaptors that sort a number of biologically important transmembrane proteins into clathrin-coated vesicles. Knockout and knockdown studies to determine GGA function are confounded by the fact that there are 3 GGA genes in mammalian cells. Thus Drosophila melanogaster is a useful model system to study tissue expression profiles and knockdown phenotypes as there is a single GGA ortholog.GGAs (Drosophila and show that there is >3-fold higher expression of GGA in male flies relative to female flies. In female flies the majority of GGA expression is in the head. In male flies GGA is not only expressed at high levels in the head but there is a gender specific increased expression which is due to the abundant expression of GGA in the testes. Using a highly specific antibody we have localised endogenous GGA protein in testes squashes, and visualised it in somatic and germ line cells. We show that GGA is expressed during multiple stages of sperm development, and co-stains with a marker of the trans-Golgi Network. This is most striking at the acroblast of early spermatids. In spite of the high expression of GGA in testes, knocking down its expression by >95% using transgenic RNAi fly lines did not affect male fertility. Therefore spermatogenesis in the male flies appears to progress normally with <5% GGA, most likely because alternative adaptors may be able to substitute partially or completely for the function of GGA. We also identify 'cueball' as a novel cargo for GGA, and mutants of cueball have been shown to have a male sterility phenotype.Here we have quantified protein expression in Drosophila we have uncovered a potential role for GGA in the testes of male flies. The gender specific higher expression of GGA, its specific enrichment in testes and its localisation to developing spermatocytes and at the acroblast of spermatids supports a role for GGA function in Drosophila spermatogenesis, even though spermatogenesis still occurs when GGA expression is depleted to <5% of control.In  Golgi-localised, γ-ear containing, ADP ribosylation factor-binding) and AP (adaptor protein)-1 are both clathrin adaptor proteins that function in the intracellular sorting of a number of biologically important transmembrane proteins between the Golgi and endocytic pathway. Where the GGAs are monomeric adaptors, AP-1 is a heterotetramer of 4 subunits , which is the functional equivalent of CIMPR, and like CIMPR contains both GGA and AP-1 clathrin adaptor sorting motifs . At the protein level GGA3 expression has been shown to vary dramatically between different human tissues with expression of both isoforms (long and short) of GGA3 in the brain [Drosophila, a search of FlyAtlas (http://flybase.org/) reveals that GGA message is found ubiquitously, and at nearly all stages of larval and adult development. To date, there is no information regarding the tissue distribution of GGA protein expression in Drosophila.The functional inter-relationship of GGAs and AP-1 has been actively debated. Both have been localised to the trans-Golgi Network (TGN) and endosomal membranes. AP-1 was shown to contain a binding site for GGAs, and this suggested the possibility that the role of GGAs may be to 'hand-on' cargo to AP-1 . However or AP-1  suggests or AP-1  or μ1A [ or AP-1  in mice iability . For GGAiability , and Symhe brain . In addihe brain ,17, thouDrosophila, and show that both GGA and AP-1 are highly expressed in Drosophila testes. In testes squashes both GGA and AP-1 co-localise with a marker of the TGN, most strikingly at the acroblast in developing spermatids. We also investigate GGA function in spermatogenesis using RNAi transgenic flies.Here we show that there is a gender specific higher expression of GGA in male Drosophila GGA and AP-1, which allowed us to localise both endogenous adaptors in Drosophila tissue culture cells [In a previous study we raised antibodies against re cells . Here weWestern blots from whole fly lysates demonstrated that GGA was expressed at higher levels in male flies than in female flies Figure , where ADrosophila testes are informative as they exhibit all stages in sperm development in a relatively simple structure. In dissected testes squashes the different stages in sperm development can be readily identified under phase contrast  rather than one interconnected ribbon found in mammalian cells. One exception is the acroblast in 'onion stage' spermatids, which is composed of a ribbon of numerous Golgi elements that forms the acrosome in mature sperm. Therefore the identity of the large puncta that are dispersed throughout the cytoplasm in spermatocytes and label for GM130, AP-1 and GGA are likely to be Golgi mini stacks.Given the abundance of GGA and AP-1 in testes as determined by Western blotting we decided to investigate their role in the testes by localising GGA and AP-1 in developing sperm. t Figure  with chat Figure . We labet Figure , GM130  flies and observed no differences in the ability to produce progeny, neither in rate, number or overall development of the progeny (results not shown). We also analysed the testes from both control Figure  and GGA Drosophila [Given the enrichment of GGA in testes we wanted to investigate what cargo proteins might require GGA for their sorting. We therefore constructed a database of cytoplasmic tails of type I transmembrane proteins and searched for those that contain a GGA binding consensus Table . Of the osophila . Of the osophila . By BLASosophila . Likewisosophila , and canIn summary, we have shown that we can efficiently deplete GGA expression to >95% in germ line derived developing sperm, and the knockdown male flies have morphologically normal testes and they exhibit no loss of fertility. Therefore with regard to fertility male flies appear to be able to function normally in the presence of <5% GGA. In addition, we have also shown that the male flies express >3 fold higher expression of GGA relative to the females, and that GGA is abundantly expressed not only in the head of flies but also specifically in the testes in multiple stages during the process of spermatogenesis. We have also identified cueball as a novel binding partner of GGA, and this protein has important functions in the growth of primary spermatocytes. These results suggest that GGA plays a role at the TGN in multiple stages of sperm development.Drosophila not only provides us with a well studied model system but functional redundancy is no longer a confounding factor as there is only a single GGA ortholog. A search on FlyAtlas reveals that GGA mRNA message is found ubiquitously, and that there is moderate message at all stages of development in nearly all larval and adult fly stages. Here we have quantified GGA protein levels and show that in female flies the majority of GGA expression is in the heads (87%), suggesting that in the females the principal function of GGA is in the head. More significantly we show that the male flies express >3 fold the total level of GGA than the females, and the increase in expression is largely confined to the testes. These results suggest the possibility of two separate roles for GGA, one in the head, where the function is shared between female and male flies, and one function in the testes that is specific to the male flies. For this paper we chose to focus our attention on the role of GGA in male testes. Interestingly, the publically available information on SymAtlas suggests that there is abundant GGA1 message in testes. Given the similarities between spermatogenesis in mammals and Drosophila it would be interesting to determine if there is gender specific higher GGA1 protein expression in mammals.The GGAs were first described in 2000 and since then our understanding of their role has risen exponentially. In mammalian cells the mRNA expression profiles of GGA1-3 predict that they are ubiquitously expressed , SymAtla,17. HoweIn order to determine where GGA may be functioning in testes we performed localisation studies in germ line and somatic cells. All antibody labelling was difficult to resolve in early spermatogonia, but GGA labelling was readily observed in Golgi mini stacks in spermatocytes. Spermatocytes are significantly larger than spermatogonia, which probably make it easier to resolve the labelling, but this is also a stage when expression of many proteins involved in spermatogenesis is switched on or upregulated. We also observed the localisation of GGA at the cone-shaped acroblast of 'onion stage' spermatids. For AP-1, despite the similar levels of overall protein expression levels in males and females, like GGA, there is proportionally more AP-1 in the male abdomens compared to the females. This expression is confined to the testes, and localises to the TGN in spermatocytes and 'onion stage' spermatids. This suggests that AP-1, like GGA, may also play a role at the TGN in different stages of sperm development. Efficient vesicle traffic is necessary in order to support the massive increase in cell membrane surface area which is required for the extensive membrane remodelling to enlarge spermatocytes (25 times increase in volume), to elongate differentiating spermatids (100-fold increase in length), and the final step of individualising spermatids. It is therefore likely that membrane trafficking may play multiple important roles in these massive remodelling events. Indeed, clathrin .We attempted to further elucidate the role of GGA in  the TGN . Therefos lethal ,12), howDrosophila is LERP [The expression and localisation of GGA and AP-1 in germ line cells supports a potential role for these adaptors in the sorting of cargo out of the TGN during spermatogenesis. What cargo might GGA be responsible for sorting in testes? As yet the only known cargo for GGA in  is LERP , which i is LERP ,7. Howev is LERP . By BLAS is LERP . Likewis is LERP . TherefoDrosophila we have uncovered a role for GGA in the testes of male flies. The gender specific higher expression of GGA, its enrichment specifically in testes, and its localisation in spermatocytes and particularly at the acroblast of developing spermatids supports a role for GGA in Drosophila spermatogenesis. We describe for the first time the knockdown of GGA in a multicellular organism and show that in terms of male fertility the flies appear to be able to function with only <5% of GGA . Since spermatogenesis is remarkably similar in mammals as it is in Drosophila, it will be of interest to determine if GGAs (particularly GGA1) play a role in mammalian spermatogenesis.In Drosophila GGA and AP-1 were raised in-house and have been described elsewhere [Antibodies against lsewhere . Other alsewhere , anti-dGDmel\Gga CG3002) RNAi flies were obtained from the Vienna Drosophila RNAi Center (http://stockcenter.vdrc.at). The library comprises 22,247 transgenic Drosophila strains with 88% coverage of the Drosophila genome [w1118. The GGA RNAi transgene was constructed by cloning a short gene fragment as an inverted repeat, and expressed using the UAS-GAL4 system in order to enable conditional inactivation of gene function in specific tissues. Two GGA transgene fly lines were established in which the insertion sites were mapped to chromosome 2 ('viable') and chromosome 3 . All controls were done in an isogenic background, and ubiquitous gene inactivation was induced using a Tubulin-Gal4 driver obtained from Bloomington Stock Center (http://flystocks.bio.indiana.edu/). Fly stocks were raised and maintained at 25°C and grown on standard cornmeal-yeast-glucose medium.GGA  and each of the two testes placed in a 3 μl drop of PBS at opposite corners of an 18 × 18 mm cover slip. The testes were carefully cut using a sharp tungsten needle to release contents, sandwiched between a microscope slide and then flash frozen in liquid nitrogen. The cover slips were removed and then the slides rapidly fixed in ice cold methanol for 5 mins at -20°C, and then acetone for 1 mins at -20°C. The samples were rehydrated in PBS 1% Triton X-100 for 10 mins, and blocked in PBS-BSA (1% BSA) for 45 mins and then labelled with primary antibody containing Hoechst  under a 22 × 22 mm cover slip at 4°C in a humid chamber overnight. The slides were washed with PBS, and then incubated with appropriate secondary antibodies diluted in PBS-BSA for 1 h at room temperature. Finally slides were washed in PBS and mounted in mounting media and imaged with a Zeiss Axiovert 200 inverted microscope using a Zeiss Plan Achromat 63x oil immersion objective, a Hamamatsu ORCA-ER2 camera, and Improvision OPENLAB software.For Western blotting, flies were separated into males and females  frozen on dry ice and then mechanically homogenised in 10 μl/fly RIPA buffer  using a microfuge pestle. The sample was sonicated, spun at 13,000 g for 5 mins and then 4x sample buffer added.For immunoprecipitations 80 male or female flies were lysed in PBS/0.5% NP40, precleared with Protein A-Sepharose, and then incubated with primary antibody for 1 h at 4°C, followed by Protein A-Sepharose for 45 mins at 4°C. The beads were washed repeatedly with PBS/0.5% NP40, a final wash in PBS and then eluted in mild conditions 50 mM Tris pH7.4/0.2% SDS or more harshly in 50 mM Tris pH7.4/2% SDS. The elutants were acetone precipitated and then resuspended in sample buffer. Western blots were quantified using IMAGE J software.Drosophila type I cytoplasmic tails, and identified 5 type I membrane proteins that contain a GGA binding consensus. Two of the hits are different transcripts of LERP which is the only known cargo for GGA in Drosophila and serves to validate the search criteria and the database construction, one has unknown function (CG12576), one protein is predicted to have lipid transporter activity (CG31150), and the most interesting hit is 'cueball' (CG12086) which shares homology to the family of LRP proteins and contains a dual GGA and adaptor sorting motif. According to FlyAtlas all 5 are expressed ubiquitously, with expression in testes. We have previously shown that cargo can regulate the recruitment of GGA to membrane; expression of the cytoplasmic tails of known GGA cargo proteins (CIMPR and sortilin) in a CD8-reporter construct specifically increases the recruitment of GGAs in HeLa cells [Drosophila GGA cargo in a GFP-reporter construct (GFP-LERP) similarly increases GGA recruitment in Dmel2 cells [GGAs are known to sort a number of type I membrane proteins by recognition of acidic dileucine motifs at the C-terminus of the cytoplasmic tails. We searched a publicly available database of cytoplasmic tail sequences to identify GGA cargo . Using aLa cells , and expon CG1257, one proJC performed all the fly manipulations, participated in the design of experiments and interpretation of data. JH performed all other experiments, participated in the design of experiments, the interpretation of data, and the writing of the manuscript. Both authors read and approved the final manuscript.AP-1 and GGA localisation in somatic cells of the seminal vesicle. Testes squashes were triple labelled for either AP-1 or GGA, and the TGN marker GCC88 and Hoechst. Note the coincidence of labelling between AP-1/GGA and GCC88 in large puncta dispersed throughout the cytoplasm. These structures most likely correspond to Golgi mini stacks. Bar, 20 μmClick here for fileAcroblast formation in GGA RNAi depleted cells. GGA was depleted ubiquitously using a Tubulin-Gal4 driver, and testes squashes from knockdown male flies (A) and control male flies (B) were triple labelled for GGA, the TGN marker GCC88 and Hoechst. Note that the acroblast in the 'onion stage' spermatid stage appears morphologically unaffected where GGA expression is depleted. Bar, 20 μmClick here for file


Growing evidence supports the validity of distinguishing major depressive disorder (MDD) plus a lifetime history of subthreshold hypomania (D(m)) from pure MDD in psychiatric classifications. The present study sought to estimate the proportion of individuals with D(m) that would have been included in RCTs for MDD using typical eligibility criteria, and examine the potential impact of including these participants on internal validity.Data were derived from the 2001–2002 National Epidemiological Survey on Alcohol and Related Conditions (NESARC), a national representative sample of 43,093 adults of the United States population. We examined the proportion of participants with a current diagnosis of pure MDD and D(m) that would have been eligible in clinical trials for MDD with a traditional set of eligibility criteria, and compared it with that of participants with bipolar 2 disorder if the same set of eligibility criteria was applied. We considered 4 models including different definitions of subthreshold hypomania.We found that more than 7 out of ten participants with pure MDD and with D(m) would have been excluded by at least one classical eligibility criterion. Prevalence rate of individuals with D(m) in RCTs for MDD with traditional eligibility criteria would have ranged from 7.98% to 22.59%. Overall exclusion rate of individuals with MDD plus at least 4 lifetime concomitant hypomanic probes significantly differ from those with pure MDD, whereas it was not significantly different in those with at least 2 lifetime concomitant hypomanic probes compared to those with bipolar 2 disorder.The current design of clinical trials for MDD may suffer from impaired external validity and potential impaired internal validity, due to the inclusion of a substantial proportion of individuals with subthreshold hypomania presenting with similar pattern of exclusion rates to those with bipolar 2 disorder, possibly resulting in a selection bias. The practice of evidence-based medicine is generally understood as the application to clinical care of knowledge derived from double blind, randomized placebo-controlled trials (RCTs) Major depressive disorder (MDD) is considered to be the most prevalent psychiatric disorder, with considerable functional and social impairment The recognition of subthreshold hypomania is important for different reasons, since depressed individuals with a lifetime history of subthreshold hypomania have greater rates of comorbidity than those without such a condition Eligibility criteria may preferentially impact subjects with D(m) in RCTs for MDD. Examining the prevalence of individuals with D(m) enrolled in clinical trials for MDD is required, and may help estimating the potential impact on internal validity as well as guiding eligibility criteria operationalization for future clinical trials in major depressive disorder.Because most RCTs examine separately efficacy of treatments for major depressive disorder and bipolar depression, the present study assessed the effect of applying exclusion criteria commonly used in clinical trials for major depressive disorder to a large , nationally representative of the U.S. general population sample, the National Epidemiological Survey on Alcohol and Related Conditions (NESARC). Our aims were 1) to estimate the proportion of individuals with D(m) that would have been included in RCTs for MDD using classical eligibility criteria, and 2) examine the potential impact of including these patients on internal validity of RCTs for MDD. We first determined the prevalence of D(m)  and pure MDD in the NESARC. We applied a standard set of exclusion criteria commonly used in clinical trials for MDD, using a method previously described by Blanco and colleagues in clinical trials for major depression We used the NESARC for our study because it is the largest representative survey with information on major depressive disorder in U.S. adults. By employing this large representative sample, we sought to stress the consequences of including participants with D(m) in clinical trials for MDD, resulting in a potential selection bias, within a broad public health context.The 2001–2002 NESARC is a nationally representative survey of the population of the United States conducted by the U.S. Census Bureau under the direction of the National Institute on Alcoholism and Alcohol Abuse (NIAAA), and described in detail elsewhere Lifetime and twelve-month psychiatric diagnoses were made according to the DSM-IV criteria with the Alcohol Use Disorder and Associated Disabilities Interview Schedule-DSM-IV Version (AUDADIS-IV), a valid and reliable fully structured diagnostic interview designed for use by professional interviewers who are not clinicians Lifetime and twelve-month mood disorders were diagnosed following the DSM-IV criteria, except for the requirement of symptoms assessing a mixed episode (criterion B for major depressive disorder and criterion C for hypomania). Consistent with the DSM-IV diagnosis guidelines, a major depressive episode (MDE) was diagnosed when an individual reported at least 2 weeks of persistent depressed mood or anhedonia, accompanied by a total of at least 5 of the 9 DSM-IV symptoms of MDE during the episode. Major depressive disorder (MDD) was defined as having a lifetime history of at least 1 MDE, without a lifetime history of mania or hypomania. Participants reporting a major depressive episode occurring during the year preceding the interview without any lifetime history of mania or hypomania were considered as having a current major depressive disorder (MDD). Participants with current MDD who declared “going anywhere or saw anyone to get help for low mood” during the year preceding the interview were considered as seeking treatment.Consistent with a prior research In each of the four models, participants with a current diagnosis of MDD were divided in 2 mutually exclusive subgroups as follows: 1) current pure MDD , 2) current MDD plus a lifetime history of subthreshold hypomania (D(m)). In Model 1, pure MDD was defined as having no lifetime history of hypomanic probe, whereas D(m) was defined as having at least 1 lifetime hypomanic probe screening criterion A or B for hypomania. In Model 2, pure MDD was defined as having 0 or 1 lifetime history of hypomanic probe, whereas D(m) was defined as having at least 2 lifetime concomitant hypomanic probes screening criterion A or B for hypomania. In Model 3, pure MDD was defined as having 0, 1, or 2 lifetime history of hypomanic probes, whereas D(m) was defined as having at least 3 lifetime concomitant hypomanic probes. At last, in Model 4, pure MDD was defined as having 0, 1, 2, or 3 lifetime history of hypomanic probes screening criterion A or B for hypomania, whereas D(m) was defined as having at least 4 lifetime concomitant hypomanic probes . Mood disorders were primary in the analyses .Exclusion criteria commonly used in clinical trials for major depressive disorder were applied to a sample representative of the general population, the NESARC, to examine the proportion of individuals with a current DSM-IV diagnosis of MDD that would have been eligible for a typical clinical trial. We used traditional efficacy eligibility criteria proposed by Zimmerman and colleagues We first determined the percentage  of survey participants with a current DSM-IV diagnosis of pure MDD and D(m) who would have been excluded by individually applying each exclusion criterion in clinical trials. Because individuals might have been excluded by more than one criterion, we also calculated the overall percentage of subjects who would have been excluded by the simultaneous application of all criteria. In each model, we conducted these analyses for all participants with a current DSM-IV diagnosis of pure MDD and D(m). The same criteria were applied to individuals with a current bipolar 2 depressive disorder to examine potential differences in the pattern of exclusion rates between these individuals and those with D(m). We conducted these analyses for all individuals with a current diagnosis of pure MDD, MDD plus a lifetime history of subthreshold hypomania, bipolar 2 depressive disorder, and for the subsamples of individuals who had sought treatment for depression, according to the four subthreshold bipolar-specifier diagnoses defined above.Because of the weighting and clustering used in the NESARC design, all statistical analyses were performed using the Taylor series linearization method, a design-based method implemented using SUDAAN, version 10 . Significance tests of sets of coefficients were performed using Wald chi-square tests based on design-corrected coefficient variance-covariance matrices. Statistical significance was evaluated using a two-sided design with alpha set at 0.05.Out of the 3,119 individuals reporting a twelve-month MDE, 2,334 had a major depressive disorder and 785  a bipolar depression. In the subsample of individuals who had sought treatment , 972 had a diagnosis of MDD and 387  a diagnosis of bipolar depression.The 12-month prevalence rates of DSM-IV MDD, MDD plus a lifetime history of subthreshold hypomania, and pure MDD were respectively 5.87% , 1.84%  and 4.04% , when using the less stringent bipolar-specifier diagnosis. Nearly one third  of the participants with a 12-month diagnosis of MDD reported at least one lifetime hypomanic probe.The percentage of participants currently presenting with major depressive disorder that would have been excluded by at least 1 out of the 6 traditional and available criteria in clinical trials for MDD ranged respectively from 71.32% to 71.96% in participants with pure MDD, and from 73.41% to 80.16% in those with D(m), according to the model used . This peThe criterion leading to the highest exclusion rate was respectively having a any past-year comorbid anxiety disorder for the full sample and the treatment-seeking subsample of participants with D(m) as well as for the treatment-seeking subsample of participants with pure MDD, and having an episode duration lower than 4 weeks or higher than 2 years for the full sample of individuals with pure MDD  and 4. PIn the full sample, a substantial proportion of participants with D(m) would have met inclusion criteria in RCTs with classical eligibility criteria, ranging from 7.98% (SE = 1.48)  to 22.59% (SE = 2.20) with a less stringent threshold . In the subsample of participants who had sought treatment for depression, this percentage would have rose from 9.56% (SE = 2.08) to 21.61% (SE = 3.04) according to the subthreshold bipolar-specifier diagnosis used .In the overall sample, the pattern of exclusion rates in individuals with a current diagnosis of MDD plus at least 4 lifetime concomitant hypomanic probes significantly differed from that of participants with pure MDD and was not significantly different from that of individuals with bipolar 2 disorder, except for the criterion “significant risk of suicide” which was significantly higher in participants with D(m) when using the more stringent subthreshold bipolar-specifier (Model 4). Furthermore, overall exclusion rate of participants with MDD plus at least 2 hypomanic probes was not significantly different from that of participants with bipolar 2 disorder . AlthougTo our knowledge, this is the first study attempting to estimate the proportion of adults with MDD plus a lifetime history of subthreshold hypomania (D(m)) that would have been included in clinical trials with traditional eligibility criteria for MDD. We found that the proportion of individuals with D(m) might range from 7.98% to 22.59% in the full sample, and from 9.56% to 21.61% in the treatment-seeking subsample, in typical clinical trials for MDD.Consistent with prior research Restrictive eligibility criteria used by RCTs at the cost of diminished external validity are justified to reach high internal validity Overall exclusion rate of participants with MDD plus at least 2 hypomanic probes was not significantly different from that of participants with bipolar 2 disorder both in the full sample and in the subsample of participants seeking treatment for depression. With that in mind, including a substantial proportion of individuals with a lifetime history of subthreshold hypomania might be responsible of a selection bias affecting internal validity of trials for MDD. In fact, our results reinforce the possibility that a substantial proportion of individuals with D(m) share similarities with those with bipolar 2 disorder. It was previously suggested that individuals with D(m) have poor response to antidepressants Furthermore, we found that individuals with D(m) may have greater risk for suicide compared with those with bipolar 2 disorder, when using the more stringent subthreshold bipolar-specifier (Model 4) in the full sample as well as in the treatment-seeking subsample. One possible explanation is that these participants, considered by the current psychiatric classifications as having unipolar depressive disorder, are less likely to benefit from a mood stabilizer compared to those with bipolar 2 disorder, as previously suggested Some limitations should be considered in interpreting these findings. First, we followed a methodology described by Blanco and colleagues Second, in absence of consensus subthreshold bipolar-specifier diagnosis Third, two exclusion criteria were not available in the NESARC and may theoretically have led to underestimate the proportion of participants excluded in clinical trials. For example, Zimmerman et al. a priori eligibility of participants and was based on national epidemiological data. It provides no information on individuals who actually enter those studies. In this way, we estimate an upper bound of the generalizability of clinical trials. Particularly, a substantial proportion of potential eligible individuals may be unwilling to participate Fourth, as previously indicated by Blanco and colleagues Fifth, although similar pattern of exclusion rates was observed in the treatment-seeking subsample compared to that of the full sample, no significant difference was found in overall exclusion rate between participants with D(m) and those with pure MDD. Although this result might be due to a lack of statistical power and a floor effect, it is possible that D(m) status may exert less impact on eligibility in individuals seeking treatment for depression.At last, severity and clinical significance of each disorder are determined by the AUDADIS-IV at the syndromal rather than symptom level. In addition, AUDADIS-IV reliability for diagnoses of anxiety disorders is only fair Despite these limitations, this study suggests that the current design of clinical trials for MDD suffers from impaired external validity as well as potential impaired internal validity due to the inclusion of a substantial proportion of individuals with D(m), that may differ from those with pure MDD but not from those with bipolar 2 disorder. We want to emphasize the need of assessing lifetime hypomanic symptoms in eligibility assessment for RCTs for MDD. Individuals with at least 4 lifetime concomitant hypomanic probes might be more accurately excluded from RCTs for MDD and considered as having bipolar 2 disorder, and those with at least 2 hypomanic probes should be systematically subject to a sensitivity analysis to test the robustness of trials’ results. Future studies would benefit from evaluating the influence of individuals with a lifetime history of subthreshold hypomania on placebo-controlled and antidepressants head-to-head clinical trials’ results, as well as efficacy and adverse effects of antidepressants in these patients.


There is considerable uncertainty about the optimal treatment of displaced 4-part fractures of the proximal humerus. Within the last decade, locking plate technology has been considered a breakthrough in the treatment of these complex injuries.We systematically identified and reviewed clinical studies on the benefits and harms after osteosynthesis with locking plates in displaced 4-part fractures.We included 14 studies with 374 four-part fractures. There were 10 case series, 3 retrospective observational comparative studies, 1 prospective observational comparative study, and no randomized trials. Small studies with a high risk of bias precluded reliable estimates of functional outcome. High rates of complications (16–64%) and reoperations (11–27%) were reported.The empirical foundation for the value of locking plates in displaced 4-part fractures of the proximal humerus is weak. We emphasize the need for well-conducted randomized trials and observational studies. There is considerable uncertainty about the optimal treatment of displaced 4-part fractures of the proximal humerus . Only 2 In the last decade, locking plate technology has been developed and has been heralded as a breakthrough in the treatment of fractures in osteoporotic bone . LockingSeveral authors of narrative reviews and clinical series have strongly recommended fixation of displaced 4-part fractures of the humerus with locking plates  and prodWe included clinical studies, randomized trials, comparative studies (prospective or retrospective), and case series, involving patients with displaced 4-part fractures according to We excluded studies of other fracture patterns  and studies in children (age < 18). Furthermore, case series were excluded if the number of fractures included was less than 10.We performed iterative searches of PubMed, Embase, Web of Science, and the Cochrane Library, restricting it to the years 1999–2009 (week 42). We also searched the One author scanned titles and abstracts for possibly eligible studies. 2 reviewers read the full-text version of potentially eligible studies, and decided independently on inclusion. Disagreements were solved by discussion. 2 authors independently extracted data on study characteristics and results using pre-tested forms. Disagreements were solved by discussion.For any randomized trials identified, we had planned to assess the risk of bias using the Cochrane Collaboration's tool for assessing risk of bias. For observational studies, we assumed that risk of bias was lower in the studies that fulfilled the following criteria: (1) the cohort was consecutively or randomly sampled; (2) dropouts or loss to follow-up were few (< 15%); (3) we considered the classification procedure to be adequate; (4) outcome was assessed blind; (5) there were no conflicts of interest; (6) we considered the cohort to be fairly representative of a typical patient with a 4-part fracture. Case series were not assessed according to methodological quality.We defined a “prospective, observational, comparative study” to be any study that collected data prospectively and compared outcomes in patients treated with plates and patients treated with a control intervention. A “retrospective, observational, comparative study” was similarly defined as a study in which outcomes were collected retrospectively, for example based on a clinical database. Furthermore, we defined a case series to be study of the outcome in a series of patients treated with locking plate, but with no comparison with patients who had received a control intervention.The search yielded 1,008 references . The majThe risk of bias in the observational, comparative studies was considerable . No studIn 2 other case series were designed to assess the effect of angulation  or type 9 companies that provided locking plates for fractures of the proximal humerus were contacted by e-mail in order to identify additional studies or to obtain unpublished data. 3 replies were received, but no additional studies or data were provided.We found that the clinical studies assessing the effect of locking plate osteosynthesis in displaced 4-part fractures were few and small, with a considerable risk of bias. No randomized trials were identified. We did note, however, that complications and reoperations after locking plate synthesis were frequent.The strength of our review is its comprehensive scope, including all major types of clinical investigations, and its thorough search strategy. The weakness is that the empirical clinical studies on this topic were scarce and not reliable. In addition, our assessment of the risk of bias is based on common sense—and not on empirical investigations—so it should be regarded as tentative. Furthermore, we excluded studies with pooled outcome data from 3- and 4-part fractures. This does not appear to have had a major influence on our results, however. We are unaware of any previously published systematic review of a similar nature. A systematic review from 2009 analyzed the benefits and harms of locking plates in 2-, 3-, and 4-part fractures . The autWe did not include studies using the AO classification system exclusively. The observational studies and the case series included in the present review showed that the use of locking screws in rigid plates has led to a new entity of complications in the treatment of proximal humeral fractures. The major harm was primary and secondary screw penetration. Primary (intraoperative) screw penetration of the humeral head is usually caused by poor surgical technique, and makes the construct prone to failure . SecondaThe severity of the injury is related to the risk of avascular necrosis, which is considered a major complication after fracture of the proximal humerus, often resulting in painful dysfunction of the shoulder. Traditional plating has been associated with a high rate of avascular necrosis, and recent publications have also reported high rates of avascular necrosis after locking plate osteosynthesis . InteresIn a recent review , an avasPrimary hemiarthroplasty has been recommended for the treatment of displaced 4-part fractures of the proximal humerus for more than 3 decades . A satisThe natural course of displaced 4-part fractures is unknown. Only a few, small series have been published, with conflicting conclusions . HoweverIn summary, there have been few clinical studies evaluating locking plate osteosynthesis in displaced 4-part fractures. Those published were small and had a high risk of bias. This precluded reliable estimates of benefits and harms, but we did note high complication rates and reoperation rates. The balance between benefits and harms is difficult to establish without further studies being done with low risk of bias. Thus, we cannot recommend the routine use of locking plate osteosynthesis for displaced 4-part fractures of the proximal humerus.