Dynamic FMT imaging (multiple times from 0 to 24 h) performed in live mice after the injection of four different FR/NIR-labeled agents, including immunoglobulin, 20–50 nm nanoparticles, a large vascular imaging agent, and a small molecule integrin antagonist, showed clear differences in the percentage of injected dose per gram of tissue (%ID/g) in liver, kidney, and bladder signal. Nanoparticles and IgG1 favored liver over kidney signal, the small molecule integrin-binding agent favored rapid kidney and bladder clearance, and the vascular agent, showed both liver and kidney clearance. Further assessment of the volume of distribution of these agents by fluorescent volume added information regarding their biodistribution and highlighted the relatively poor extravasation into tissue by IgG1. These studies demonstrate the ability of quantitative FMT imaging of FR/NIR agents to non-invasively visualize and quantify the biodistribution of different agents over time.When small molecules or proteins are injected into live animals, their physical and chemical properties will significantly affect pharmacokinetics, tissue penetration, and the ultimate routes of metabolism and clearance. Fluorescence molecular tomography (FMT) offers the ability to non-invasively image and quantify temporal changes in fluorescence throughout the major organ systems of living animals, in a manner analogous to traditional approaches with radiolabeled agents. This approach is best used with biotherapeutics  or large-scaffold drug-delivery vectors, that are minimally affected by low-level fluorophore conjugation. Application to small molecule drugs should take into account the significant impact of fluorophore labeling on size and physicochemical properties, however, the presents studies show that this technique is readily applied to small molecule agents developed for far-red (FR) or near infrared (NIR) imaging. Quantification by non-invasive FMT correlated well with both fluorescence from tissue homogenates as well as with planar (2D) fluorescence reflectance imaging of excised intact organs (r The type of agent, dictated by physicochemical properties such as size, charge, and chemical composition, will not only influence pharmacokinetics and biodistribution but will also influence the volume of distribution, the route of metabolism, and clearance. In the development of both therapeutic agents and imaging agents, all of these parameters contribute to the risk assessment of an agent with regard to efficiency of targeting, assessment of off-target accumulation, and prediction of potential sites of adverse reactions.Pharmacokinetic, biodistribution, and metabolic clearance characteristics are important properties that can influence the overall efficacy of novel therapeutics, imaging agents, and macromolecular delivery vectors Preclinical biodistribution and pharmacokinetics data for investigational agents are routinely obtained in animal studies using radiolabeled materials ex vivo assessments of organs, both by 2D organ imaging as well as by quantification of fluorescence within tissue homogenates. Subsequent dynamic FMT imaging (multiple time points from 0 to 24 h) was performed using four different types of FR/NIR-labeled imaging agents, including a 1.4 kDa drug-like integrin antagonist In these studies, we built upon recent advances in optical tomographic imaging and FR/NIR agents to demonstrate the utility of non-invasive FMT to detect, localize, and quantify agent biodistribution in the major organ systems. For this, we used empirical testing with a variety of FR/NIR-labeled agents to establish 3D regions of interest (ROI) that optimally capture different organs with minimal overlap. BSA-VT680XL FMT results agreed well with comprehensive FMT biodistribution assessment, by virtue of its safety and ease of use, may be ideal for the routine study of small molecule imaging agents or larger therapeutic biomolecules . Future combination of FMT with CT imaging or fluorescent contrast agents, to provide anatomical co-registration, may further advance the robustness of biodistribution imaging.To quantitate fluorescence biodistribution in the different organs of living mice, and changes over time, studies were designed as depicted in In the case of stomach, bladder, kidney, and heart imaging, the ROIs placed to define these organs were deliberately sized larger than the actual organs to provide sufficient room for mouse-to-mouse variability. Liver, intestine, and lung ROIs were sized as a compromise to achieve maximal specific organ capture and minimize overlap with other organ systems. The composite ROI model for FMT imaging of organ tissue biodistribution is represented in Three mice per group were administered an IV injection of 2 nmol of BSA-VT680XL and imaged non-invasively by FMT2500 at 24 h to reconstruct 3-dimensional fluorescence images  using weex vivo assessments of organs, both by FRI organ imaging as well as by quantification of fluorescence within tissue homogenates. FRI images of the post-mortem tissues , showing three patterns of dynamic changes over time for the four agents. Little information could be obtained from this data alone with regard to the kinetics of extravasation of these agents into the tissue. However, comparison to the typical plasma pharmacokinetic profiles for these agents (1/2>100 h). IgG1 showed the slowest extravasation into tissue (peaks at 24 h) and also has a tissue half-life >100 h.Dynamic FMT imaging (multiple time points from 0 to 24 h) was performed using four different FR- or NIR-labeled agents, including IgG1 (150 kDa), fluorescent nanoparticles (20–50 nm diameter), a vascular imaging agent (250 kDa), and an integrin antagonist (1.4 kDa). These agents differ widely in molecular weight and chemical composition and would be expected also to differ in pharmacokinetics, tissue distribution, tissue clearance kinetics, and routes of metabolism. Careful, quantitative interpretation of temporal imaging datasets, taking into account the signal contributed by circulating agent, is an important consideration when determining the amount and time course of agent extravasation and accumulation in specific organ tissues, as illustrated in e agents , normalie agents , quantifi.e. subtraction of normalized PK curves from imaging data) was applied to each organ system, again making the assumption that at time zero all of the signal can be attributed to the blood. The plasma PK values were normalized for each organ and subtracted from each organ’s total %ID/g value . The resulting plasma corrected tissue levels gave a clear picture of the kinetics of extravasation of each agent into each organ tissue. Bladder signal was not corrected because we have found minimal vascular contribution to bladder fluorescence.Further analysis of whole body imaging datasets for the four agents represented in i.e. less than 5–6%ID/g) seen in other tissues. The vascular and nanoparticle agents , it has been a useful pharmacokinetic parameter to predict the extent of distribution into bodily water or body fat. FMT quantification of fluorescence volume (mm3) in living mice would, in theory, provide a more direct surrogate for volume of distribution calculations, and results for four fluorescent agents are shown in i.e. the increase in apparent voxel numbers when signal occurs on the interface between multiple voxels in 3 dimensions). This effect would be expected to be particularly strong when applied to imaging tissue as fine and widespread as the vascular system, and it is interesting to note that all four agents showed approximately 7000–8000 mm3 of total fluorescence volume at time zero, the time at which all fluorescence would be presumed to be in the vasculature. Assuming that a 25 g mouse has approximately 2 mm3 of blood, this starting fluorescence volume corresponds to an approximate 4-fold overestimate of blood volume.By standard pharmaceutical calculations, the volume of distribution , is the theoretical volume of fluid into which an administered drug would have to be diluted to produce the resulting plasma drug levels. Although volume of distribution has no absolute physiological meaning  and compared to approximate standard measures for vascular volume (∼2 mm3), total body water (∼18 mm3), and total body fat (∼4 mm3) in a 25 g mouse For IgG1, the volume of fluorescence changed little over time (∼0% increase), with the loss of plasma signal balanced by liver tissue accumulation . The relNon-invasive preclinical imaging techniques are quickly becoming important tools in biomedical research, leading to significant advances in the ability to monitor disease progression and the impact of therapeutic intervention in a variety of disease areas. With the recent advances in NIR tomographic imaging technology we have been able to demonstrate the capabilities of fluorescence molecular tomographic imaging to detect and quantify fluorescent biomarkers of disease in sites of cancer, inflammation, and infectious disease 18F or 64Cu. The seamless integration of tomographic FMT and PET datasets showed excellent spatial co-registration and quantitative correlation, supporting the utility of FMT imaging as a non-radioactive surrogate for PET imaging in preclinical studies. To further take advantage of the robustness of FMT imaging, and the use of fluorescence rather than radioactivity, the present studies support the expansion of the horizons of preclinical FMT imaging into quantitative biodistribution assessment. FMT imaging of fluorescent agents of vastly different characteristics, from small molecule to large proteins to nanoparticles, yielded quantitative time course information regarding differential kinetics, organ distributions, and clearance routes. By incorporating parallel blood pharmacokinetic data into the imaging analysis, we were further able to separately quantify the vascular signal and the extravasated tissue signal. This analysis, although not the standard for PET biodistribution studies (which does not remove the blood contribution), provided useful information regarding the rate of extravasation into tissues and was particularly helpful in understanding biodistribution patterns at very early time points at which agents remained high in circulation. Subtracting circulating fluorescence from tissue fluorescence was also critical in the proper interpretation of biodistribution results in some instances; without PK correction, skin, heart, and lung distribution were often grossly overestimated due to the relatively high contribution of the circulating signal to the overall imaging results. Additional techniques are being explored that may allow the selective analysis of vascular and tissue signal without the need for parallel pharmacokinetic studies, further streamlining the biodistribution imaging process. Other readouts, such as liver:kidney ratios and fluorescence volume of distribution will need extensive validation to establish their utility as robust readouts.Recent studies by Nahrendorf et al. [30] have further advanced the level of validation of FMT imaging by performing hybrid PET-FMT imaging studies, using targeted agents labeled both with a NIR fluorophore and The major utility of FMT technology in biodistribution imaging is likely to be the application to the design and modification of antibodies or large protein therapeutics, as well as to the characterization of novel optical imaging agents of any size. Such an approach, in many instances, could be an option when rapid biodistribution screening is needed. Fluorescence-based tomographic imaging by FMT uses no radioactivity, minimally affects performance of large proteins, and presents an easy and user-friendly option with no need for special safety considerations or animal quarantine. Fluorescence approaches, however, may be less useful than PET or SPECT imaging in the study of very low molecular weight drug biodistribution, due to the significant impact on function, pharmacokinetics, and biodistribution when you double or triple a drug’s size by fluorophore conjugation.Although disease site fluorescence has been approached carefully in many published studies, little work has been done to provide the full picture of agent accumulation in non-target tissues other than comparisons to specific control sites. With the current capabilities of FMT imaging, we are now examining agent targeting to sites of disease in the context of general biodistribution, helping in the overall understanding of both the disease biology and specificity of agent deposition. Further addition of kinetic readouts can be added to understand rates of agent deposition to specific sites (in the case of imaging time points post agent injection) and to track the progression of disease (when imaging at different times following initiation of disease). Multi-channel capability for FMT imaging also allows the simultaneous imaging of multiple agents, further decreasing the need for large cohorts of animals.ex vivo assessment of organ tissue signal, provides a powerful tool for quantifying the distribution and metabolic fate of FR- and NIR-labeled agents. FMT imaging in antibody and imaging agent research, utilizing NIR fluorescent labels, will provide a useful, non-invasive tool for understanding their distribution and metabolism.In conclusion, we have demonstrated the ability of the FMT imaging to non-invasively visualize and quantify whole-body tissue biodistribution in a robust and consistent manner. The consistency of the quantitative tomography, as well as its excellent correlation with All experiments were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol (#01-0904) was approved by PerkinElmer’s (In Vivo Imaging Division) IACUC guidelines for animal care and use. No invasive or surgical procedures were used in these studies, but all imaging activities were performed under appropriate anesthesia to minimize animal distress.ad libitum. For PK studies, specific pathogen-free female CD-1 mice (6–8 weeks of age) were obtained from the same vendor and kept under the same conditions as above.For whole body biodistribution studies, specific pathogen-free female Nu/Nu mice (4–6 weeks of age) were obtained from Charles River  and housed in a controlled environment  under specific-pathogen free conditions with water and food provided Commercially available  near infrared imaging agents and custom conjugates were used to optimize and perform biodistribution studies  that was purchased from Sigma-Aldrich , and murine IgG1 purchased from Rockland  to provide reference agents for biodistribution studies. N-hydroxysuccinimide (NHS)-ester labeling of proteins was performed using the 680 nm fluorophore . Briefly, the NHS ester moieties of VivoTag 680XL (pH 7 for 1 h at room temperature) reacted with primary amino groups of proteins to form stable amide bonds. In this manner, a small number of lysine residues  in antibodies and other proteins can be labeled with fluorophore, with free fluorophore removed by size exclusion chromatography. Conjugation ratios were easily assessed by measuring absorbance of the conjugates at 280 and 680 nm with appropriate extinction coefficients to determine protein and fluorophore concentration, respectively. Labeling for both BSA (BSA-VT680XL) and IgG1 (IgG1-VT680XL) achieved ∼3 fluorophores per protein molecule, and these conjugates were dosed in animals at 2 nmoles of fluorophore/mouse IV. These conjugates showed excellent stability when stored at 4C and, upon in vivo imaging system . For whole body imaging, the anesthetized mice were placed in the supine position, centrally in the imaging cassette, to capture the whole body (excluding the head) of the animal within the imaging scan field of the imaging system. For imaging the head, the mice were placed in the prone position in the imaging cassette for a separate scan. After positioning the mouse, the imaging cassette was adjusted to the proper depth to gently restrain the mouse and then inserted into the heated docking system (regulated at ∼37°C) in the FMT imaging chamber. A FR/NIR laser diode transilluminated  the animal body, with signal detection occurring via a thermoelectrically cooled CCD camera placed on the opposite side of the imaged animal. Appropriate optical filters allowed the collection of both fluorescence and excitation datasets, and the multiple source-detector fluorescence projections were normalized to the paired collection of laser excitation data. The entire image acquisition sequence took approximately ∼8–10 min (torso) and ∼2–3 min (head) per mouse.Nu/Nu mice were injected IV with 2 nmoles of FR/NIR fluorescent agents and imaged at time 0, 1, 3, 6, and 24 h following injection. At the time of imaging, mice were anesthetized by inhalation in a chamber containing a mixture of isoflorane and oxygen then imaged using the FMT 2500 fluorescence molecular tomography The collected fluorescence data was reconstructed by FMT 2500 system software  which compensates for the effects of tissue heterogeneity on light scattering To determine the %ID/g in the different organs of living mice without the contribution from circulating blood, pharmacokinetic data was used to subtract blood levels from each organ at each time point. This was done by assuming that all organ signal at the first imaging time was due to fluorescence within the blood vessels of the organ as depicted in in vivo, they were sacrificed by carbon dioxide asphyxiation. The organs  were removed post-mortem then imaged by fluorescence reflectance imaging using FMT 2500’s planar imaging capability and frozen at −80 C. For some animals, organs were also subjected to a homogenization procedure. The organ tissues were thawed in the dark, and then weighed. The appropriate volume of PBS (0.5 mL/mg of tissue) was added to each tissue prior to homogenization to an even consistency. Planar fluorescence reflectance imaging was used to measure the fluorescence from serial dilutions of each homogenate (10 uL/sample) to assure quantification was performed within ranges of fluorophore concentration not subject to intermolecular fluorescence quenching. Appropriate dilutions of normal tissue homogenates were used to measure background fluorescence which was subtracted from the final calculations.After animals were imaged CD-1 or nude mice were injected IV with a known amount of fluorogenic agent. Terminal blood samples were collected by cardiac puncture from each mouse at the appropriate time point following carbon dioxide asphyxiation. A minimum of two mice were sampled for each time point. Plasma was collected by centrifugation and diluted 1∶2 in DMSO to assure capture of maximal fluorescent signal. Plasma fluorescence was measured using a fluorescence plate reader . The data was normalized to a standard curved prepared with known concentrations of the agent to quantify blood levels in ug/ml, and results were converted to percent of injected dose. Mouse inter-strain differences in pharmacokinetic profiles were well within the range of variability seen within mouse strains.


Over the last 60 years, the spotlight of research has periodically returned to the cerebellum as new techniques and insights have emerged. Because of its simple homogeneous structure, limited diversity of cell types and characteristic behavioral pathologies, the cerebellum is a natural home for studies of cell specification, patterning, and neuronal migration. However, recent evidence has extended the traditional range of perceived cerebellar function to include modulation of cognitive processes and implicated cerebellar hypoplasia and Purkinje neuron hypo-cellularity with autistic spectrum disorder. In the light of this emerging frontier, we review the key stages and genetic mechanisms behind cerebellum development. In particular, we discuss the role of the midbrain hindbrain isthmic organizer in the development of the cerebellar vermis and the specification and differentiation of Purkinje cells and granule neurons. These developmental processes are then considered in relation to recent insights into selected human developmental cerebellar defects: Joubert syndrome, Dandy–Walker malformation, and pontocerebellar hypoplasia. Finally, we review current research that opens up the possibility of using the mouse as a genetic model to study the role of the cerebellum in cognitive function. The cerebellum is an intriguing component of the central nervous system. From one perspective it is a famously simple neuronanatomical circuit constructed from a relatively few neuronal types and comprising a single uniform microarchitecture . HoweverDespite the uniformity of its cellular structure, the cerebellum is divided into clear anatomical divisions on the basis of a transverse fissures that separate lobes. These are folds in what is a continuous ribbon of neural circuitry that, in humans, would extend over a meter in anteroposterior length . A primaThese three major functional subdivisions of the cerebellum have been long been recognized and used to calibrate defects in developmental morphogenesis, many of which, as described below, have a prominent affect on the vermis (spinocerebellum). However, it should be clear from the above that functional consequences of developmental disorders may also depend on the degree of disruption to the formation of cerebellar nuclei and the precision of their inputs, which are difficult to assess. In addition, appreciation of cerebellar dysfunction is colored by the simple constraint that the cerebellum is best-known and understood in terms of its integration of proprioceptive information in the control of movement . TypicalHowever, recent studies have highlighted the possibility that cerebellar defects might underlie some of the symptoms in subsets of patients diagnosed with neurodevelopmental disorders like autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD), and schizophrenia . As thesThese advances are coupled with an increasing potential to interpret both anatomical and genetic phenotypes in terms of specific aspects of cerebellar development. This has been driven by substantial progress in understanding the origins of different cerebellar cell types and their interactions within the last 10 years. Fate-maps based on the genetic identity of different cell types, a molecular dissection of their interactions and new anatomical techniques to trace long range connections in the brain  have revAs the classification of brain pathologies with cerebellar involvement and known genetic associations have been reviewed extensively , whereas the position of the IsO at the mid-hindbrain boundary is determined the mutually repressive activities of the homeobox genes Otx2 (orthodenticle homeobox 2) anteriorly, and Gbx2 (gastrulation brain homeobox 2), posteriorly . The FGF and WNT signaling pathways are prime examples. Since the initial identification of Fgf8 and Wnt1 gene expression in cells at the IsO /r1 region. The analysis of embryos homozygous for hypomorphic alleles of Fgf8, suggested that the maintenance of normal levels of FGF8 signaling was particularly important for the formation of medial cerebellar tissue . A study by the Joyner lab has shown that the developmental stage at which Fgf8 expression is disrupted is a key determinant of the severity of vermis hypoplasia; Fgf8 deletion from the early (pre-E9.5) IsO cause severe vermis hypoplasia, whereas only mild hypoplasia in the anterior vermis resulted from Fgf8 deletion between E9.5 and E11  essentially represent phenocopies of FGF hypomorphic cerebella, by displaying a specific loss of the cerebellar vermis  form, suggesting that this defect was primarily caused by the failure to generate GLI3R . However, recent studies have provided evidence for important roles for  the IsO . Recentlte GLI3R .An important prediction of these studies in the mouse is that many human cerebellar disorders with strong cerebellar vermis involvement are likely to be caused by the disruption of IsO function during the earliest stages of cerebellar development. This proposition will be discussed further in Section “Mechanistic Insights into the Causes of Human Cerebellar Defects” in the context of recent findings on the mechanisms underlying human cerebellar malformations.Figures B and Figure 2A). Evidence that the production of different neuronal lineages is spatially restricted during cerebellar development comes from loss-of-function and lineage tracing experiments in the mouse. Atoh1 , a gene specifically expressed in the RL, resulted in the failure to form an external germinal layer (EGL) and EGL-derived granule cells .After initial patterning and growth of r1 to form the cerebellar anlage, neurogenesis is initiated in two distinct germinal centers, the ventricular zone (VZ) and rhombic lip (RL). All cerebellar neurons and glia as well as progenitors that populate a number of extracerebellar nuclei are born within these germinal zones . In particular, these discoveries also point to a time window of sensitivity to developmental damage that might target deep cerebellar nuclei but leave later born granule cell derivatives untouched. Such a window of potential vulnerability to intrinsic or extrinsic damage to the embryo might have selective effects on cerebellar function (in particular connectivity) that are not necessarily correlated with substantial reduction in cerebellar size.In addition to GCps, the  neurons . Moreove neurons . This raVentricular zone. Genetic fate-mapping of cells in the cerebellar VZ, demonstrated that all GABA-ergic neurons, including Purkinje, Golgi, basket, and stellate cells, as well as small GABA-ergic neurons of the deep cerebellar nuclei are derived from this region . Compared to the number of glutamatergic granule neurons in the adult cerebellum, the contribution of GABA-ergic neurons to the over-all size of the cerebellum is relatively minor. Thus, defects in the generation of GABA-ergic neurons are not expected to result directly in significant cerebellar hypoplasia. However, as we discuss in the next section, VZ-derived Purkinje cell progenitors are the primary source of mitogen to GCps in the EGL. Thus, the absence or mislocalization of Purkinje cells due to VZ defects could be responsible for cerebellar hypoplasia owing to a deficit in GCp proliferation and postnatal cerebellar growth . Indeed, Ptf1a and Atoh1. Atoh1 results in RL cells entering the roof plate, indicating that ATOH1 activity suppresses the adoption of this non-neuronal fate. This is reminiscent of the mutual inhibitory interactions that specify progenitor domains within the spinal cord . In addition, cell migration defects resulting in the ectopic localization of Purkinje cells are likely to underlie many examples of cerebellar heterotopias  or SHH signal transduction components like Smo (smoothened), Gli1, and Gli2 from GCps have all been shown to result in defects in GCp proliferation and cerebellar hypoplasia  proliferation of GCps in the vermis and hemispheres equally, resulting in a phenotype that differs significantly from early IsO defects with disproportionally hypoplastic vermis.Apc (adenomatous polyposis coli) gene or stabilization of β-catenin in GCps inhibited their proliferation and enhanced their differentiation. As a consequence, these mice exhibited cerebellar hypoplasia and ataxia. The Wechsler-Reya group confirmed the potent inhibition of GCp proliferation and cerebellar hypoplasia as a result of hyperactivation of β-catenin in GCps, but found additionally that the proliferation of GABA-ergic progenitors in the VZ was significantly increased when signaling was increased in this progenitor zone . WNT/β-catenin signaling is also active at later stages of cerebellar development, particularly in the germinal zones and Bergmann glia . The roltor zone . The obstor zone , in agreSimilar complexities have been observed upon altering FGF signaling during cerebellar development. Increased FGF signaling upon deletion of Sprouty genes, which encode FGF antagonists, have opposite effects on cerebellar growth depending on the time of deletion. Upregulated FGF signaling during early development is associated with the expansion of vermis progenitors in anterior r1 and an expansion of the width of the vermis. Increased FGF signaling during late embryonic and postnatal stages on the other hand is associated with abnormalities in Bergmann glial and Purkinje cell differentiation, reduced SHH production by these cells as well as an inhibitory effect on the responsiveness of GCps to SHH. As a consequence of these changes, GCps prematurely exit the cell cycle and differentiate, leading to general cerebellar hypoplasia .in vitro cultures have to be interpreted with considerable caution. Although these studies are useful as initial proof-of-principle studies, validation and analysis in the appropriate cell types in an intact developing cerebellum are essential.These observations provide a window on some of the complexities of understanding the role of particular pathways in cerebellar development. Not only does one pathway have multiple functions at different developmental time points, the effects of deregulated signaling in one neuronal progenitor subpopulation can be vastly different from another. In the light of these observations, any attempt to infer mechanistic explanations for cerebellar phenotypes from observations made in unrelated cell types or simplified Insights gained from studies in model organisms have greatly improved our ability to identify the underlying processes responsible for many congenital cerebellar anomalies that affect the human population. In this section, we aim to demonstrate some of these new insights by discussing our current understanding of two major syndromes associated with cerebellar hypoplasia: Joubert syndrome and Dandy–Walker malformation. We contrast the remarkable progress that has been made in our understanding of these syndromes with pontocerebellar hypoplasia, a group of conditions for which candidate genes have been identified but little mechanistic insights are available.Joubert syndrome is characterized by cerebellar vermis hypoplasia and abnormal superior cerebellar peduncles and associated with both movement disorders and mental retardation . JoubertAHI1 (Abelson helper integration site 1). First, they showed convincingly that vermis hypoplasia in Ahi1-/- mouse mutants is associated with an expanded roof plate and a vermis midline “fusion” defect  and roof plate expansion at the expense of the expansion of vermis progenitors has been shown to take place in FGF pathway mutants. However, WNT signaling was diminished in the medial cerebellar anlage at E13.5, which correlated with reduced proliferation of vermis progenitors adjacent to the midline. Finally, WNT/β-catenin activation by LiCl treatment at E12.5 and E13.5 partially rescued the proliferation defect and midline “fusion,” confirming reduced WNT signaling as an important pathogenic mechanism in Joubert syndrome  mutants, the data suggest that a WNT signaling defects might also underlie Joubert syndrome caused by mutations in Joubert-associated genes other than AHI1. This study clearly implicates deregulated WNT signaling as a mechanism in Joubert syndrome.Despite the wealth of human genetics data and direct links to ciliary assembly and function, the cilia-dependent developmental pathways disrupted in Joubert syndrome are poorly understood. A recent study by the Gleeson group provided important insights into the causes of cerebellar phenotypes in Joubert syndrome caused by mutations in ” defect . This cepomorphs . From stpomorphs . WNT sigsyndrome . A similTmem67 (transmembrane protein 67) in the regulation of SHH and WNT signaling. The authors report a significant expansion of the fourth ventricle roof plate in Tmem67-/- embryos, a phenotype consistent with a disruption of early IsO function and vermis hypoplasia/aplasia. Based on the studies discussed so far, one might predict that reduced WNT signaling in these mutants may lead directly to reduced activity of the IsO, early loss of vermis progenitors, or reduced proliferation of cells at the midline, akin to the hypothesis to explain vermis defects in Ahi-/- embryos. Indeed, the authors present data to suggest that WNT/β-catenin signaling is reduced in Tmem67-/- embryonic fibroblasts from embryos with Joubert syndrome-like phenotypes. They also report reduced Shh expression in the ventral neural tube. It will be important to examine WNT and SHH signaling in mes/r1 in Tmem67 mutants, in order to draw direct conclusions as to the involvement of these pathways in cerebellar development at these critical early developmental stages. Taken together, these recent studies suggest that several signaling pathways might be affected as a result of defective ciliary function and that the cerebellar vermis phenotype in Joubert syndrome is due to the deregulation of signaling pathways required for early IsO function and vermis expansion.Tmem216 (transmembrane protein 216), a gene mutated in Joubert and Meckel syndromes, resulted in ciliogenesis defects, and impaired centrosome docking to cilia due to the mislocalization of hyperactivated RhoA. Disheveled1 (Dvl1) phosphorylation in response to Tmem216 knockdown and mild PCP phenotypes were observed in zebrafish  pathway has also been implicated in the pathogenesis of Joubert syndrome. Knockdown or mutation of ebrafish . The rolKAL1  was excluded by Ueno et al. in their study, leaving the possibility that mutations in other Kallmann syndrome-associated genes that affect FGF signaling might be responsible.Although the critical roles of FGF signaling during cerebellar development are well-established in model organisms, FGF signaling defects have not been linked directly to cerebellar vermis defects in humans. This might be due to the fact that FGF hypomorphic mutations that reduce FGF signaling sufficiently to cause a cerebellar vermis phenotype, are also detrimental to the development of many other organs, such that foetuses carrying these mutations will not survive past birth. Two groups have reported rare patients with Kallmann syndrome and partial cerebellar vermis a/hypoplasia, initially diagnosed as Dandy–Walker malformation . MutatioZIC1 (zinc finger protein of the cerebellum 1) and ZIC4 (zinc finger protein of the cerebellum 4) genes on 3q24 as candidate genes, an observation since confirmed by other groups  mouse mutants after the identification of FOXC1 as a candidate gene for Dandy–Walker malformation in humans, has revealed an novel mechanism whereby cerebellar vermis hypoplasia could arise. MouseFoxc1 hypomorphs exhibit vermis hypoplasia confirming that reduced FOXC1 function is responsible for Dandy–Walker malformation in humans. Intriguingly, Foxc1 is not expressed in the developing cerebellum, but in mesenchymal tissue of the posterior fossa covering the cerebellar anlage from about E11.5. The IsO, roof plate, and RL initially develop normally in these mutants, but Atoh1 expression in GCps in the medial EGL was lost by E14.5 of development. As Atoh1 is required for GCp proliferation  gene that encodes chromatin modifying protein 1A (CHMP1A), has been identified as a candidate gene for pontocerebellar hypoplasia. Patient-derived lymphoblastoid cell lines showed reduced proliferation and increased expression of the cell cycle inhibitor INK4A , a target of the Polycomb group member BMI1 (B lymphoma Mo-MLV insertion region 1 homolog). This change in expression correlated with reduced BMI1 recruitment to an Ink4a regulatory region, suggesting that CHMP1A may regulate Polycomb recruitment to gene loci, thereby phenocopying the cerebellar phenotype in Bmi1-/- mutants and , EXOSC3 (exosome component 3), RARS2 (arginyl-tRNA synthetase 2), TSEN54 (tRNA-splicing endonuclease subunit 54), TSEN2 (tRNA-splicing endonuclease subunit 2), and TSEN34 . Furthermore, and contrary to prevailing theories  mutant mouse is a particularly significant step in linking cerebellar defects to ASD (Tsc1 only from cerebellar Purkinje cells during postnatal development, Tsc1 mutant mice. These mice scored low on sociability and social novelty tests when presented with the opportunity to interact with a new mouse. Although communication and language are not easily assessed in mice, pups will attempt to attract the mother’s attention by ultrasonic vocalizations when separated from her. Tsc1 mutant pups showed an increase in these vocalizations, suggesting that the lack of Tsc1 in the cerebellum can affect this process, although the relevance of this observation to ASDs is not clear. Finally, Tsc1-deficient mice exhibited excessive grooming, an indication of repetitive behaviors and demonstrated cognitive inflexibility in a reversal learning paradigm, perhaps indicative of an insistence of sameness (Despite the potential complexities of cerebellar origins of ASD, a recent analysis of an ASD-like condition in a tuberous sclerosis complex 1 (s to ASD . Firstlysameness . This laThe identification of the genetic defects responsible for specific anatomical abnormalities in the cerebellum is likely to continue at an unrivalled speed owing largely to the revolution in next generation sequencing. However, it should be remembered that knowledge of the genetic basis of disease is only the first step in understanding the condition. The next big challenge is to unravel the developmental and molecular mechanisms by which genetic changes manifest in a particular disease phenotype. Studies in model systems like the mouse have been essential in this quest and will no doubt continue to remain so. Without the fundamental knowledge gained from these experimental studies, translation of genetic findings to preventative and curative strategies will not be possible.Unraveling the salient features and functional importance of cerebellar connectivity with cortical regions implicated in psychiatric conditions like autism, ADHD and schizophrenia, is of the utmost importance of we want to understand the involvement of the cerebellum in these conditions . Multi-dSignificant differences between the neuroanatomy of rodents and primates may emerge as barriers to understanding cognitive deficits in the mouse, despite all its advantages as an experimental and genetic model. Nevertheless, we highlight here that recent insights gained in the Tsc1 mutant suggest that the substrates for cortico-cerebellar modulation of behavior are present in the rodent. While this field is relatively undeveloped, the promise for understanding the full range of cerebellum function through exploration of these systems in mouse presents a research avenue of enormous promise.Finally, this article almost exclusively focuses on cerebellar hypoplasia. Many other congenital cerebellar malformations have been described, including a range of conditions that appear to be associated with malformation of the posterior fossa and cerebellar overgrowth. For example, rasopathies like Costello syndrome are characterized by Chiari type I malformation and cerebellar tonsillar herniation . One migThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The G-protein linked signaling system (GPLS) comprises a large number of G-proteins, G protein-coupled receptors (GPCRs), GPCR ligands, and downstream effector molecules. G-proteins interact with both GPCRs and downstream effectors such as cyclic adenosine monophosphate (cAMP), phosphatidylinositols, and ion channels. The GPLS is implicated in the pathophysiology and pharmacology of both major depressive disorder (MDD) and bipolar disorder (BPD). This study evaluated whether GPLS is altered at the transcript level. The gene expression in the dorsolateral prefrontal (DLPFC) and anterior cingulate (ACC) were compared from MDD, BPD, and control subjects using Affymetrix Gene Chips and real time quantitative PCR. High quality brain tissue was used in the study to control for confounding effects of agonal events, tissue pH, RNA integrity, gender, and age. GPLS signaling transcripts were altered especially in the ACC of BPD and MDD subjects. Transcript levels of molecules which repress cAMP activity were increased in BPD and decreased in MDD. Two orphan GPCRs, GPRC5B and GPR37, showed significantly decreased expression levels in MDD, and significantly increased expression levels in BPD. Our results suggest opposite changes in BPD and MDD in the GPLS, “activated” cAMP signaling activity in BPD and “blunted” cAMP signaling activity in MDD. GPRC5B and GPR37 both appear to have behavioral effects, and are also candidate genes for neurodegenerative disorders. In the context of the opposite changes observed in BPD and MDD, these GPCRs warrant further study of their brain effects. The G-protein linked signaling system (GPLS) comprises G-proteins, G protein-coupled receptors (GPCRs), GPCR ligands, and downstream effector molecules. G-proteins interact with both GPCRs and downstream effectors such as cyclic adenosine monophosphate (cAMP), phosphatidylinositols, and ion channels. The GPLS is implicated in the pathophysiology and pharmacology of both major depressive disorder (MDD) and bipolar disorder (BPD). GPCRs are integral membrane proteins with key roles in numerous physiological cellular processes. GPCRs can signal ligand binding events by conformational changes that activate intracellular G proteins. Active G proteins in turn trigger intracellular downstream signaling pathways. Evidence suggests that altered GPCRs and their two major downstream signaling pathways, mediated by effectors such as cAMP and phosphatidylinositol, may be involved in the pathophysiology of BPD and MDD as well as in the mechanism of drug treatment for these disorders. Altered immunoreactivities and functional activities of proteins related to cAMP signaling pathways have been reported in the brain and peripheral blood cells of BPD patients , and nine patients with MDD (Cohort A). The MDD results were validated with an additional independent cohort, including seven controls and five MDD patients (Cohort B). Toxicology data and/or recent clinical data were examined for the BPD and MDD subjects and detailed clinical information is summarized in Table Brain tissue and clinical information were obtained by the UCI Brain Bank staff with the informed consent of decedents' next-of-kin, and were processed through a standard protocol approved by the local UCI Institutional Review Board  were found to incorrectly BLAST to other transcripts, a refined CDF based on an UniGene cluster was developed  between experimental duplicates were considered to be reliably measured genes, and were subjected to downstream analyses. These genes were analyzed in a mixed-model ANOVA utilizing Partek Pro 6.0  for the main effect of the diagnostic classification  and for sex .The hypergeometric distribution was used for calculating probabilities that the observed number of genes in each gene category for cAMP or PI signaling pathways (listed in Table t) values for the target transcripts were normalized to averaged Ct values for two reference transcripts, Jagged 1 (JAG1) and solute carrier family 9 isoform 1 (SLC9A1), which showed equivalent expression levels among the three diagnostic groups  throughout the 3 brain regions on our normalized microarray data. After DNase digestion and purification, total RNA (1 μg) samples from each of the 34 subjects in Cohorts A and B were used as a template for first-strand cDNA synthesis using poly-dT primer. The mRNA for each transcript was measured using the SybrGreen system with Prism model 7000-sequence detection instrument  and primer sequences listed in Table t, which correlatedinversely with the target mRNA concentration, was measured as the cycle number at which the SybrGreen fluorescent signal increased abovea pre-set threshold level. The qPCR experiments were performed three times for each transcript, and the Ct triplicate values were averaged.For further technical evaluation of the microarray data, we evaluated mRNA expression levels by quantitative real-time reverse transcriptase PCR (qPCR) for the seven genes listed in Table in situ hybridization (ISH) using the ACC regions from six representative subjects to evaluate the cellular localization of GPRC5B and GPR37 mRNA expression. A formal statistical analysis of the ISH data was not applied due to low subject number. ISH was performed following a protocol as described elsewhere  using 35S-labeled sense RNA probes for GPRC5B and GPR37, and (2) pre-treating tissue with RNase A before hybridizing with the antisense probes.The gene expression fold changes for GPRC5B and GPR37 showed an increase in BPD and decrease in MDD. We selected those genes for p < 0.05) in either Cohort A or B and either mood disorder, BPD or MDD, are listed in Table p-values > 0.1) suggesting that residual pH-agonal effects on the expression levels of GPCR genes did not remain in the data set, even though complete elimination of these factors may not be possible with matching of subject's age and pH, and inclusion of subjects that had rapid deaths.Significantly dysregulated GPLS genes that passed FDR . Thus, there was a nominal enrichment of genes that act negatively on cAMP signaling activity  in BPD in the ACC.The genes involved in cAMP signaling pathways, which were differentially expressed in ACC of BPD patients compared to controls, are listed in Table p > 0.05).In contrast, of those genes that repressed cAMP signaling activity in MDD Figure  there wai/G12-linked EDG2 was increased in ACC of BPD, while Gi-linked EDG1 was decreased in ACC of MDD. EDG1 and EDG2 regulate neuronal and glial functions, including neurite retraction, neurogenesis, and axonal guidance  were not altered in BPD and MDD compared with controls in our study, which is consistent with a previous study .The phosphatidylinositol (PI) signaling pathway related genes which were differentially expressed in ACC of BPD patients are shown in Figure The PI signaling pathway related genes which were differentially expressed in ACC of MDD patients compared with controls are summarized in Figure q-linked monoamine GPCRs , which was consistent with previous microarray studies  and G protein-coupled receptor 37 (GPR37)as shown in Table We followed up on results primarily that met FDR in cohort A for BPD, and both Cohorts A and B for MDD, except for RGS20, which we followed up based upon Cohort A meeting FDR while Cohort B met uncorrected p-value significance only. Nine GPLS transcripts were selected for validation, five for BPD and four for MDD. The qPCR results showed 100% concordance between qPCR and microarray fold change for BPD in ACC Table  and 4/5 GPR37 mRNA was preferentially expressed in subcortical white matter. The sense probe hybridization signal showed background signal. GPR37 mRNA expression in subcortical white matter was higher in ACC of both of the BPD subjects compared to both of the control subjects, whereas GPR37 mRNA expression in subcortical white matter was lower in ACC of both of the MDD subjects compared to both of the control subjects Figure . This prTranscriptional levels of molecules acting as negative regulators in cAMP signaling pathways are increased in BPD and decreased in MDD, which may compensate for the activated cAMP signaling activity in BPD and the blunted cAMP signaling activity in MDD,q and PLC-beta in the disorder,Expression of PRKCB1, ITPR1, INPP5F, and ITPKB were altered in MDD, which may reflect functional impairments of GExpression of INPP1 and phosphatidylinositol 3-kinases were altered in BPD suggesting that these molecules may be involved in both the pathophysiology and the mechanism(s) of action of lithium treatment of BPD, andTwo orphan GPCR genes, GPRC5B and GPR37, showed significant increases in ACC of BPD, and decreases in ACC and DLPFC of MDD.The results of the study of GPLS transcriptional profiling showed differences in BPD and MDD in both the cAMP- and phosphatidylinositol signaling pathways. The following pattern of alterations in GPLS were observed:Among all GPCRs, the most consistent differential expression patterns were observed for GPRC5B and GPR37. GPRC5B was significantly increased in ACC and DLPFC of BPD, while significantly decreased in ACC and DLPFC of MDD patients. GPR37 was significantly increased in ACC of BPD, and significantly decreased in ACC and DLPFC of MDD patients. The consistent cortical alterations of GPRC5B and GPR37 expression observed in MDD patients suggest a role of these genes in the pathophysiology of MDD. So far, these two orphan GPCRs have not been highlighted as candidate susceptible genes for BPD and MDD, however previous microarray data supports decrease of both GPRC5B and GPR37 expressions in MDD patients (Aston et al., −/− mice shows reduced striatal dopamine content and enhanced amphetamine sensitivity (Marazziti et al., GPR37 appears to facilitate dopamine neurotransmission, and reductions in brain GPR37 leads to behavioral alterations in conditioned place preferences to reinforcements such as cocaine or amphetamine (Marazziti et al., GPRC5B is phylogenetically classified into a type C GPCR, however, in contrast to other type C genes, GPRC5B has a short N terminal domain (Brauner-Osborne and Krogsgaard-Larsen, The expression patterns of GPRC5B and GPR37 suggest that both of these orphan GPCRs may be involved in various cellular functional activities in the brain including neuronal and glial cells. Functional characterization of GPRC5B and GPR37 may provide new insights into the pathophysiology of mood disorder, and potential novel strategies for developing methods for diagnoses and treatment of the disorders.There are limitations to this post-mortem study of GPLS pathway expression. We conducted seven qPCR validations of genes that passed FDR, and although a small number, those genes did represent large enough percent fold changes to validate by qPCR. Other technologies could be used that are less costly, such as Nanostring or Fluidigm for additional validation studies, which are now being conducted on larger sample sizes. In addition, we do not have confirmed toxicological analysis of all drugs of abuse and therapeutic levels to rule out these effects in explaining case—control expression differences. Finally, we are aware that small residual agonal effects might exist that are not corrected in this analysis, however we did confirm that the present data presented in Table This study highlights transcript profile differences in GPLS pathways in both BPD and MDD. Since sample sizes were low, we also used a second independent cohort in the case of MDD gene expression to check concordance. We relied upon duplicate microarray analysis at independent laboratories, and used gene expression values that were significantly correlated between sites, and employed FDR to select genes to advance for qPCR experimental validations.The disturbances observed in cAMP- and phosphatidylinositol- pathway regulation may be due to differential activation of individual GPCRs and individual differences in ligand- receptor sensitivity, suggesting that functional and genetic characterization of these candidate genes may provide further understanding of the pathophysiology of mood disorders. Further studies will also be required to investigate additional GPCR signaling pathway features such as receptor phosphorylation, beta-arrestin recruitment, receptor desensitization, and internalization, and beta-gamma mediated signaling. The results of the present study suggest that future functional activity studies of the GPLS pathways would elucidate the relationship of the observed transcript changes to protein levels and functional outcomes in mood disorders.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Genome-wide gene expression profile using deep sequencing technologies can drive the discovery of cancer biomarkers and therapeutic targets. Such efforts are often limited to profiling the expression signature of either mRNA or microRNA (miRNA) in a single type of cancer.Here we provided an integrated analysis of the genome-wide mRNA and miRNA expression profiles of three different genitourinary cancers: carcinomas of the bladder, kidney and testis.Our results highlight the general or cancer-specific roles of several genes and miRNAs that may serve as candidate oncogenes or suppressors of tumor development. Further comparative analyses at the systems level revealed that significant aberrations of the cell adhesion process, p53 signaling, calcium signaling, the ECM-receptor and cell cycle pathways, the DNA repair and replication processes and the immune and inflammatory response processes were the common hallmarks of human cancers. Gene sets showing testicular cancer-specific deregulation patterns were mainly implicated in processes related to male reproductive function, and general disruptions of multiple metabolic pathways and processes related to cell migration were the characteristic molecular events for renal and bladder cancer, respectively. Furthermore, we also demonstrated that tumors with the same histological origins and genes with similar functions tended to group together in a clustering analysis. By assessing the correlation between the expression of each miRNA and its targets, we determined that deregulation of ‘key’ miRNAs may result in the global aberration of one or more pathways or processes as a whole.This systematic analysis deciphered the molecular phenotypes of three genitourinary cancers and investigated their variations at the miRNA level simultaneously. Our results provided a valuable source for future studies and highlighted some promising genes, miRNAs, pathways and processes that may be useful for diagnostic or therapeutic applications. Genitourinary neoplasms pose a large burden on human healthcare, with prostatic carcinoma ranked as the most predominant genitourinary tumors followed by bladder and kidney carcinoma in America Despite intensive efforts by many investigators over the past years, the exact mechanisms involved in the initiation and progression of these genitourinary cancers remain largely unclear. Transitional cell carcinoma (TCC) of the bladder is the most common (∼90%) histopathological type of bladder cancer. Many factors, including chromosomal anomalies, genetic polymorphisms, and genetic and epigenetic alterations, are thought to contribute to the tumorigenesis or progression of TCC Our previous work on ccRCC demonstrated that simultaneously profiling the expression patterns of mRNAs and miRNAs in the same panel of cancer patients using a massively parallel sequencing platform (Illumina GA II) is a highly integrative and reproducible way of dissecting the molecular basis of human cancer 2 ratio ≥1, FDR≤0.01) in TGCT, TCC and ccRCC, respectively. The numbers of miRNAs significantly deregulated in those three cancers were 254, 226 and 118, respectively . Statistical methods for determining the differentially expressed genes and miRNAs are described previously Large numbers of transcripts showed consistent patterns of expression among the patients  with the same genitourinary cancer. Compared to the matched normal control, a total of 3181, 4321 and 4596 mRNAs were significantly differentially expressed  in deregulated genes in TGCT, TCC and ccRCC are listed in All of the genes that were deregulated in each of the three cancers were used as inputs for the DAVID bioinformatics resource separately to identify the significantly deregulated pathways P≤0.05). The detailed results of the GO enrichment analysis are listed in P≤5.0×10-05). Other important biological processes that were significantly enriched in genes deregulated in any two of the three cancers included the developmental process, cell adhesion and the cell differentiation process, which were affected in TGCT and TCC; the immune and inflammatory response process, which was affected in TGCT and ccRCC; and the DNA replication and repair processes, which was affected in TCC and ccRCC. Remarkably, we also observed that gene sets showing TGCT-specific deregulation patterns were mainly implicated in processes related to male reproductive function, such as spermatogenesis, fertilization and spermatid development Gene sets showing the same deregulated expression patterns in multiple cancers or genes showing cancer-specific expression deregulation are thought to be functionally important to the tumorigenic processes 2 ratio ≥1, FDR ≤0.01) in at least 75% of the 27 patients. As shown in We next studied the differences in gene expression profiles that could be used to distinguish between, or give biological insights into the molecular disparities of the tumors with distinct histological origins. Of the 596 genes that were deregulated in all the three cancers , 64 wereIn this comparative study, we observed that over half (17/31) of the miRNAs that were aberrantly expressed in all the three cancers exhibited consistent expression patterns . Many ofAs shown in P≤0.05) were found between 77, 196 and 444 miRNA-mRNA pairs in TGCT, TCC and ccRCC, respectively  MAP7, which is predominantly expressed in cells of epithelial origin and is able to stabilize microtubules, was observed to be up-regulated in TCC  By careful examination of the individual miRNAs that negatively correlated with the expression levels of their target genes, we found that miR-367 (belonging to the miR-302-367 cluster mentioned above) was up-regulated solely in TGCT and its predicted target P = 0.037) and three of the targets  participate in regulating the JAK-STAT signaling pathway. However, no enriched biological process was found in the correlated target gene sets of miR-30c and miR-363.As expected, the expression levels of some miRNAs were significantly correlated with multiple target genes. 43 predicted target genes under the regulation of the up-regulated miR-19a/b were all down-regulated in TCC, and 11 of them were predicted to be regulated by both miR-19a and miR-19b . In addiThe present study provided an integrated overview of three genitourinary cancers based on their whole-genome expression profiles of both mRNA and miRNA. All matched tumor-normal sample pairs from the 27 cancer patients were prepared and sequenced using the same protocols on the same platform, and the resulting raw sequencing data were also submitted to similar preliminary analyses Research over recent decades has suggested that transformation of normal cells into malignant cancers may be governed by several common rules Systematic comparisons of three genitourinary cancers also demonstrated that a large number of deregulated genes were enriched in specific pathways or processes that characterize the distinct biological behaviors of each cancer. Gene sets solely deregulated in TGCT are mainly implicated in male reproduction-related processes such as spermatogenesis, spermatid development and fertilization, indicating that normal testicular function had been lost during the conversion of normal somatic cells into tumor cells. Meanwhile, global down-regulation of multiple metabolic pathways and significant deregulation of biological processes related to blood coagulation, lipid and ion transport were only observed in ccRCC. As reported previously, disorder of intravascular coagulation may result in renal lesions CCND1, PERP and RRM2TCF19 encodes a transcription factor that is assumed to play a pivotal role in regulating the expression of target genes. The gene product of CCNB1 (cyclin B1) and cdc2 form the maturation-promoting complex, which is important for stimulating mitosis CENPF may play a role in chromosome segregation during mitosis FAM107A was identified in the three genitourinary tumors DCN (decorin) is capable of suppressing the growth of various tumor cells TCF21 was aberrantly methylated and silenced in head and neck squamous cell carcinoma and non-small-cell lung cancer C2orf40 was hypermethylated and transcriptional silenced in colorectal carcinoma and glioma, and its over-expression led to a significant decrease in cell growth TCF19) in cluster 1 and 2 may also be responsible for important functions in these three cancers and thus are worthy of further attention in future studies.Classification of cancer based on the expression patterns of key molecular markers that can be used for cancer diagnosis, prognosis and prediction had been widely described and applied in previous studies CNN1 is a late stage differentiation marker of smooth muscle cells, and a decrease in CNN1 is associated with underdeveloped renal tumor vessels that lack integrity AMOTL1, CAV1 and COL15A1) in this class were functionally associated with the angiogenesis process. AMOTL1 controls cell migration and cell-cell adhesion in vivo during angiogenesis CAV1 enhances the formation of endothelial capillaries in vitro and in vivoCAV1 in ccRCC had been documented previously and is thought to be important for the progression of ccRCC In addition, we also identified a subset of genes (cluster 3) that were up-regulated in ccRCC but down-regulated in TCC and TGCT. The expression of miRNAs are an important class of small non-coding RNAs that are capable of regulating the temporal and tissue-specific expression patterns of protein coding genes at the post-transcriptional level, blocking translation and/or leading to mRNA degradation ARRDC3 can bind and degrade the ITGβ4 protein, which affects the proliferation, migration and invasion of breast cancer cells in vitroCBX7, decreases with malignancy grade and neoplasia stage in thyroid cancer JAK1, CCND2 and SPRED1) participating in JAK-STAT signaling. Down-regulation of miR-20b may therefore lead to the activation of the JAK-STAT pathway, which has been documented in many cancer studies P = 0.037), which may, in part, account for the characteristic phenotype of ccRCC . All tumor and matched normal adjacent tissues in this study were obtained from the biobank (CXC201005260001A) of the Urinogenital Cancer Genomics Consortium (UCGC) in China. Information on the 27 patients  analyzed is summarized in 2 ratio using the normalized TPM (transcripts per million reads) values. Subsequently, we performed a rigorous significance test to determine the differentially expressed genes and miRNAs P-values (P) were corrected for multiple tests using the FDR  adjustments Total RNA was extracted from normal adjacent tissues and tumor tissues of each patients studied. Four and five micrograms of total RNA were subjected to mRNA and miRNA sequencing library preparation, respectively, before they were sequenced using the Illumina GAII platform according to manufacturer's instructions . Manipulation of raw sequencing data, reference alignment and determination of differentially expressed mRNA and miRNA were all performed as described previously 2 ratio ≥1, FDR ≤0.01) in at least 75% of 27 patients. The uncentered correlation was calculated to cluster genes and tumor samples based on the relative expression of these 64 genes using the average linkage hierarchical clustering algorithm applied in the cluster program Of the 596 genes that were deregulated in all three cancers, only 64 genes were differentially expressed  to determine whether the expression levels of each miRNA and its mRNA targets were negatively correlated (P≤0.05). Finally, the putative targets confirmed by the correlation analysis were subjected to further analysis.We investigated the biological relevance of differentially expressed miRNAs through their regulation on target genes. First, a putative target gene set was generated according to the intersect result of any two predicted algorithms of DIANA-microT 3.0 P-values from modified Fisher's exact test; more information can be found in http://david.abcc.ncifcrf.gov/) were deemed to be statistically significantly deviated from the expected distribution, and thus, the corresponding pathways were enriched with the deregulated genes in question. Considering the large amount and complex branch structure of GO biological processes, we used a significance threshold P-value adjusted by Benjamini of 0.05 for biological process terms to control the false discovery rate Different sets of differentially expressed genes and miRNA targets were used as input for the DAVID bioinformatics resource. KEGG pathways with EASE values ≤0.05 Click here for additional data file.Table S1Differentially expressed genes in TGCT, TCC and ccRCC.(XLS)Click here for additional data file.Table S2Differentially expressed miRNAs in TGCT, TCC and ccRCC.(XLS)Click here for additional data file.Table S3KEGG pathways enriched in differentially expressed genes in TGCT, TCC and ccRCC.(XLS)Click here for additional data file.Table S4Biological process analysis of gene sets that were deregulated uniformly in all cancers or only in TCC, TGCT or ccRCC.(XLS)Click here for additional data file.Table S5Genes deregulated commonly in TCC, TGCT and ccRCC.(XLS)Click here for additional data file.Table S6Target prediction of differentially expressed miRNA in TGCT, TCC and ccRCC.(XLS)Click here for additional data file.Table S7Biological processes involvement of target genes of differentially expressed miRNAs.(XLS)Click here for additional data file.Table S8Clinical information on the 27 patients in our study.(XLS)Click here for additional data file.


We also explored the extent to which insulin resistance (IR) is a distinctive feature of NAFLD. To answer these questions, we used systems biology approaches, such as gene enrichment analysis, protein–protein interaction networks, and gene prioritization, based on multi-level data extracted by computational data mining. We observed that the leading disease pathways associated with NAFLD did not significantly differ from those of AFLD. However, systems biology revealed the importance of each molecular process behind each of the two diseases, and dissected distinctive molecular NAFLD and AFLD-signatures. Comparative co-analysis of NAFLD and AFLD clarified the participation of NAFLD, but not AFLD, in cardiovascular disease, and showed that insulin signaling is impaired in fatty liver regardless of the noxa, but the putative regulatory mechanisms associated with NAFLD seem to encompass a complex network of genes and proteins, plausible of epigenetic modifications. Gene prioritization showed a cancer-related functional map that suggests that the fatty transformation of the liver tissue is regardless of the cause, an emerging mechanism of ubiquitous oncogenic activation. In conclusion, similar underlying disease mechanisms lead to NAFLD and AFLD, but specific ones depict a particular disease signature that has a different impact on the systemic context.The abnormal accumulation of fat in the liver is often related either to metabolic risk factors associated with metabolic syndrome in the absence of alcohol consumption  or to chronic alcohol consumption . Clinical and histological studies suggest that NAFLD and AFLD share pathogenic mechanisms. Nevertheless, current data are still inconclusive as to whether the underlying biological process and disease pathways of NAFLD and AFLD are alike. Our primary aim was to integrate  Despite the fact that the causative PNPLA3, also known as adiponutrin), is critically involved in the genetic susceptibility of fatty liver in both NAFLD Interestingly, advances in genome analysis have shown that rs738409 C/G, a nonsynonymous coding (I148M) gene variant located in human patatin-like phospholipase domain containing 3 gene  has significantly changed our understanding about the pathogenesis of complex diseases.The omics data are growing exponentially, sometimes negatively impacting our capacity of extracting and interpreting biological insights from them. Fortunately, interesting computational resources have been developed, such as data-mining techniques, which not only help us in assembling information from the biomedical literature, but are also devoted to uncover details that are of practical value for reveling disease pathogenesis.Nevertheless, the multi-level high-throughput Hence, our primary aim was to integrate genomic, molecular, and physiological data about NAFLD and AFLD to answer the question of whether both the diseases share the same underlying pathogenic mechanisms. In addition, we explored the biological processes and associated-disease pathways behind NAFLD and AFLD to evaluate the extent to which IR is (or is not) a distinctive feature between them.To answer these questions, we used data mining and systems biology approaches, such as a gene enrichment analysis and protein–protein interaction networks.To assemble the available evidence about NAFLD- and AFLD-associated biological processes in a systematic manner, we used the text mining platform PESCADOR (Platform for Exploration of Significant Concepts Associated to co-Occurrence Relationships) Table S1) and 314 terms were found for NAFLD (Table S2).Thus, with the queries “alcoholic AND (steatosis OR fatty liver) NOT ” for AFLD and “nonalcoholic OR non-alcoholic AND fatty liver OR steatosis” for NAFLD, we retrieved 823 papers and 1345 co-occurrences for AFLD, and 868 papers and 2217 co-occurrences for NAFLD. The query involves retrieving extensible markup language (XML) PubMed abstracts for PMID list, passing XML PubMed abstracts for NLPROT analysis , and tagging protein names and performing co-occurrences analyses. After carrying out terms' tagging, a total of 228 gene/protein terms were identified for AFLD  tool ToppGene Suite (http://toppgene.cchmc.org) and ToppCluster (http://toppcluster.cchmc.org), which could detect functional enrichment of the candidate genes/proteins list based on Transcriptome, Proteome, Regulome (TFBS and miRNA), Ontologies , Phenotype (human disease and mouse phenotype), Pharmacome (Drug-Gene associations), literature co-citation, and other features Based on the list of genes/proteins identified as explained earlier, we decided to explore the interactions between them in an integrative fashion to provide a “functional molecular map” of both the clinical disorders. Thus, functional enrichment analysis was performed by the bioinformatic resource This application was selected because the gene functional annotations are based on a comprehensive list of databases that includes among others, annotation for drugs, disease and mouse phenotype, miRNAs, and allows the identification and prioritization of novel disease candidate genes in the interactome. Furthermore, the statistical methods for quantitative enrichment are reliable http://homes.esat.kuleuven.be/~bioiuser/endeavour/endeavour.php.In addition, we used a strategy of gene prioritization under the hypothesis that the already known associated disease loci and proteins might be useful as a template to look for unknown molecular targets involved in the pathogenesis of NAFLD and AFLD. Thus, we performed a comprehensive analysis of candidate regions generated by the freely accessible ENDEAVOUR software available at Table S1 and Table S2, were used as training genes in the Endeavour platform; subsequently, the application prioritized the entire human genome looking for similarities between the candidates and the models built with the training genes. This prioritization covers most of the aspects of knowledge available on genes and gene products  ENDEAVOUR is a software application for the computational prioritization of candidate genes underlying biological processes or diseases, based on their similarity to known genes involved in a disease as previously described IGF1R with arterial hypertension and HNF4a with type 2 diabetes after being predicted by this tool This application was selected because its system was validated experimentally by extensive leave-one-out cross-validations showing an excellent performance Figures were constructed under the graphical platforms for exploring the biological networks, MEDUSA The results of text mining for biomolecular interactions among terms associated with AFLD and NAFLD are shown in Once AFLD-associated terms and interactions were displayed graphically, a dendriform hierarchical hub appeared centered on CYP2E1, TNFα, CD14 , IL10, PPARα, and the apoptosis regulator BCLXL Figure 1Table S2), we performed a functional enrichment analysis. Interestingly, the analysis showed that AFLD-reported loci and proteins are integrated into several similar functional pathways and biological processes that did not significantly differ from those of NAFLD  molecules as well as interleukins and interleukin receptors were overrepresented in both AFLD and NAFLD, but ATP-binding cassette transporters, caspases, and acyl-CoA synthetases were only significantly predicted in NAFLD Table 1.Prediction of relatedness with mouse phenotype showed that candidate AFLD genes/proteins were significantly associated with abnormal liver and hepatobiliary system morphology, and to a lesser extent with lipid metabolism; on the other hand, candidate NAFLD gene/ proteins were significantly associated with glucose and lipid homeostasis Table 1.We further performed a multiple genes/ proteins analysis that simultaneously encompassed the candidate list of AFLD and NAFLD to obtain a functional modular map that identifies shared and specific features of each disease, including potential regulatory mechanism relationships. Cross-comparing enrichment analyses showed that NAFLD is associated with a myriad of complex pathways that include among the others, insulin signaling, caspases and mitochondrial-related apoptosis, stress induction of heat shock proteins, cellular proliferation, hypoxia induction, and protein associated with epigenetic regulation Figure 3Conversely, cross-comparing enrichment analyses showed that AFLD is associated with a more reduced network of disease pathways, mostly focused on modulation of the immune response, toll-like receptor signaling, and cytokines Figure 3Figure S1, and a functional modular map of the multiple gene/ protein analysis encompassing the candidate list of NAFLD and AFLD based on cellular component is shown in Figure S2. Of note, although there are specific genes associated with either AFLD or NAFLD, there is not a specific pathway for both of the diseases.Interestingly, cardiovascular-related pathways were more enriched in NAFLD in comparison with AFLD (platelet plug formation and endothelins), as shown in Our secondary aim was to explore whether impaired insulin signaling is a molecular process associated with both the conditions, regardless of the insult , or if IR is restricted to NAFLD and thereby to MetS.Comparative co-analysis of both the data sets showed that insulin signaling is impaired in both the liver disorders, but the biological processes and putative regulatory mechanisms associated with NAFLD seem to encompass a large interconnected network of genes and proteins, including, but not restricted to, FOXO1, SIRT1, dipeptidylpeptidase IV , PPARγ, PER, and HDAC3 Figure 4Gene prioritizations shows that fatty liver either associated with metabolic factors or alcohol is strongly associated with cancer pathways and ubiquitously malignant cellular transformationTable S3) and AFLD (Table S4), based on the top-ranked significant P-values that reflect the significance of enrichment. Then, we performed a new comparative co-analysis of both the data sets generated by ENDEAVOUR, and we explored novel putative disease pathways. Surprisingly, in addition to genes associated with IR or type 2 diabetes, i.e. HNF4A Figure S3). Moreover, growth factors associated signaling pathways involved in cell proliferation, cell migration, survival, and chemotaxis, such as IGF, PDGF or their receptors, and growth factors active in angiogenesis and endothelial cell growth, such as VEGF, were also highly enriched .We used the above-explained approach of gene prioritization, and based on the results of ENDEAVOUR output, we selected the top-ranked candidate genes prioritized for NAFLD  that might modulate AFLD, such as miR-9, or NALFD, such as miR-146a, miR-18a, and miR-22 Figure 5Figure S4). Interestingly, based on these different targets, some miRNAs, such as miR-7a and miR-146a, were once again predicted.Comparative co-analysis of both the data sets generated by ENDEAVOUR platform showed an even more complex prediction of miRNA .There is agreement about the fact that the clinical distinction between NAFLD and AFLD is often challenging, and misclassification owing to difficulties in collecting reliable information about the amount of alcohol consumption from patients is a source of bias. Likewise, the pathogenic distinction between both the disorders is not only fuzzy, but physicians agree that similar major molecular mechanisms are shared between NAFLD and AFLD, including inflammatory pathways and fibrogenesis Our primary result basically showed that the leading biological process and disease pathways associated with NAFLD did not significantly differ from those predicted for AFLD. Nevertheless, systems biology revealed the weight of each molecular process behind each of the two diseases, and dissected distinctive molecular NAFLD and AFLD signatures.Figure S2).For instance, apoptosis seems to be the common cell death process in NAFLD and AFLD, but AFLD is mainly associated with the so-called extrinsic pathway related to tumor necrosis family, which would be modulated by proapoptotic BCl2 family members. In contrast, NAFLD seems to be primary associated with FAS-induced apoptosis, which is highly interconnected to an intricate network of metabolic stressors, activation of caspases, and a collection of proteins that modulate apoptosis, necrosis, and inflammation. It is worth mentioning that changes in mitochondrial membrane permeabilization and endoplasmic reticulum stress are central features in both AFLD and NAFLD, as shown in the prediction of the cellular compartment .An integrative knowledge about the disease pathogenesis of NAFLD and ALFD will pave the way towards formulation of new hypothesis. In addition, if underlying mechanisms are common, the same future therapeutic approaches may improve both conditions. Nevertheless, more experimental data and clinical studies are needed in order to accomplish this observation.Figure S1a functional modular map of the multiple gene/ protein analysis encompassing the candidate list of NAFLD and AFLD based on functional genes. Results of functional association analysis performed by the bioinformatics resource ToppCluster (http://toppcluster.cchmc.org). Right side of the figure depicts the highly significant enrichments for sets of genes (red hexagon) of the NAFLD term list; left side of the figure depicts the highly significant enrichments for sets of genes of the AFLD term list; and the analysis of the intersection of genes and gene functions (green squares) between NAFLD and AFLD is shown in the center of the figure. The network is shown as a cytoscape graph.Graphic illustration of (TIF)Click here for additional data file.Figure S2a functional modular map of the multiple gene/ protein analysis encompassing the candidate list of NAFLD and AFLD based on cellular component. Results of functional association analysis performed by the bioinformatics resource ToppCluster (http://toppcluster.cchmc.org). Right side of the figure depicts the highly significant enrichments for cellular components (green squares) of the NAFLD-term list; left side of the figure depicts the highly significant enrichments for cellular components of the AFLD term list; and the genes (red hexagons) and analysis of the intersection between NAFLD and AFLD is shown in the center of the figure. The network is shown as a cytoscape graph.Graphic illustration of (TIF)Click here for additional data file.Figure S3Computational prioritization of candidate genes underlying NAFLD and AFLD and comparative co-analysis of genes pathways (green squares). Prioritization was done by the bioinformatic tool ENDEAVOUR, and the figure shows the results of the cluster analysis of the first top 200 prioritized candidate genes from the whole human genome (23.712 genes), with a significant association with the training set of NAFLD and AFLD. Functional association analysis was performed by the bioinformatics resource ToppCluster (http://toppcluster.cchmc.org). Right side of the figure depicts the highly significant enrichments for sets of genes (red hexagons) of the NAFLD term list; left side of the figure depicts the highly significant enrichments for sets of genes of the AFLD term list; and the analysis of the intersection of functional genes between NAFLD and AFLD is shown in the center of the figure. The network is shown as a cytoscape graph.(TIF)Click here for additional data file.Figure S4Computational prioritization of candidate genes underlying NAFLD and AFLD and comparative co-analysis of predicted miRNAs (violet squares). Prioritization was done by the bioinformatic tool ENDEAVOUR, and the figure shows the results of the cluster analysis of the first top 200 prioritized candidate genes (red squares) from the whole human genome (23.712 genes), with a significant association with the training set of NAFLD and AFLD. Functional association analysis was performed by the bioinformatics resource ToppCluster (http://toppcluster.cchmc.org). The network is shown as a cytoscape graph.(TIF)Click here for additional data file.Figure S5Computational prioritization of candidate genes underlying NAFLD and AFLD and comparative co-analysis of predicted drugs (orange squares). Prioritization was done by the bioinformatic tool ENDEAVOUR, and the figure shows the results of the cluster analysis of the first top 200 prioritized candidate genes (red squares) from the whole human genome (23.712 genes), with a significant association with the training set of NAFLD and AFLD. Functional association analysis was performed by the bioinformatics resource ToppCluster (http://toppcluster.cchmc.org). The network is shown as a cytoscape graph.(TIF)Click here for additional data file.Table S1Genes terms identified in 823 published abstracts by the PESCADOR platform (Platform for Exploration of Significant Concepts Associatedto co-Occurrence Relationships) with the query “alcoholic AND (steatosis OR fatty liver) NOT ” for AFLD.(DOC)Click here for additional data file.Table S2Genes terms identified in 868 published abstracts by the PESCADOR platform (Platform for Exploration of Significant Concepts Associatedto co-Occurrence Relationships) with the query “nonalcoholic OR non-alcoholic) AND (fatty liver OR steatosis)” for NAFLD.(DOC)Click here for additional data file.Table S3Results of gene prioritization: the top ranked candidate genes prioritized for NAFLD.(DOC)Click here for additional data file.Table S4Results of gene prioritization: the top ranked candidate genes prioritized for AFLD.(DOC)Click here for additional data file.


Cardiac function depends on the ability to switch between fatty acid and glucose oxidation for energy production in response to changes in substrate availability and energetic stress. In obese and diabetic individuals, increased reliance on fatty acids and reduced metabolic flexibility are thought to contribute to the development of cardiovascular disease. Mechanisms by which cardiac mitochondria contribute to diet-induced metabolic inflexibility were investigated. Mice were fed a high fat or low fat diet for 1 d, 1 wk, and 20 wk. Cardiac mitochondria isolated from mice fed a high fat diet displayed a diminished ability to utilize the glycolytically derived substrate pyruvate. This response was rapid, occurring within the first day on the diet, and persisted for up to 20 wk. A selective increase in the expression of pyruvate dehydrogenase kinase 4 and inhibition of pyruvate dehydrogenase are responsible for the rapid suppression of pyruvate utilization. An important consequence is that pyruvate dehydrogenase is sensitized to inhibition when mitochondria respire in the presence of fatty acids. Additionally, increased expression of pyruvate dehydrogenase kinase 4 preceded any observed diet-induced reductions in the levels of glucose transporter type 4 and glycolytic enzymes and, as judged by Akt phosphorylation, insulin signaling. Importantly, diminished insulin signaling evident at 1 wk on the high fat diet did not occur in pyruvate dehydrogenase kinase 4 knockout mice. Dietary intervention leads to a rapid decline in pyruvate dehydrogenase kinase 4 levels and recovery of pyruvate dehydrogenase activity indicating an additional form of regulation. Finally, an overnight fast elicits a metabolic response similar to that induced by high dietary fat obscuring diet-induced metabolic changes. Thus, our data indicate that diet-induced inhibition of pyruvate dehydrogenase may be an initiating event in decreased oxidation of glucose and increased reliance of the heart on fatty acids for energy production. Approximately 70% of the ATP produced in the heart is derived from the oxidation of fatty acids. The heart does, however, increase the utilization of glucose in response to enhanced availability, increased workload, and physiologic and pathophysiological stress. This metabolic flexibility is essential for cardiac function because: 1) Glucose relative to fatty acids produces more ATP per molecule of oxygen consumed and 2) Glycolytic intermediates are required for processes other than ATP production . ChronicThe mitochondrial enzyme pyruvate dehydrogenase (PDH) is a key regulatory point in glucose oxidation, catalyzing the oxidative decarboxylation of pyruvate and formation of acetyl-CoA and NADH. PDH is stringently regulated, both allosterically and posttranslationally, based on energy demands as well as glucose and fatty acid availability. Reversible phosphorylation of PDH occurs on the α-chain of the E1 subunit, one of three subunits that comprise the PDH complex. Three phosphorylation sites exist . Phosphorylation of PDH inhibits enzyme activity and is catalyzed by four isoforms of pyruvate dehydrogenase kinase, three of which are expressed in the heart . Dephosphorylation and activation of PDH is catalyzed by two pyruvate dehydrogenase phosphatases (PDP1 and PDP2) -10. Inhiin vivo. Nevertheless, it is important to consider that PDH activity exhibits dynamic changes in response to shifting energy demands and substrate availability. Thus, depending on the method and/or time of sample preparation, the active fraction of PDH may not accurately reflect diet-induced changes in PDH function. Further confounding integration of reported findings, food deprivation is well known to elicit increases in PDK4 message, protein, and/or activity  designates an alkylated cysteine. All analyses are in the positive ion mode with Q1 and Q3 resolution set to 0.7Da.(DOCX)Click here for additional data file.


Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder, characterized by painful swelling of muscles and connective tissue in the early years of life, consequently leading to ossification at a mean age of 4-5 years. We report FOP in a 2-year-old boy with palpable masses in the frontal and lower cervical paraspinal and left periscapular muscles.He was born with hallux valgus. Despite this hallmark, he was referred to the hospital with the primary diagnosis of hematoma, but further investigation indicated FOP. The patient was discharged from the hospital with non steroidal anti-inflammatory drugs (NSAID) and education of the parents. The importance of this case was that in spite of the early occurrence of the typical presentation of FOP for more than one year and the fact that the patient's mother was a physician who had consulted with many specialists, the diagnosis had been missed.This indicates that the general physicians, radiologists and other specialists' awareness and knowledge of FOP is insufficient. Fibrodysplasia ossificans progressiva (FOP) or myositis ossificans progressiva also known as Munchmeyer disease is a rare autosomal dominant condition that begins in childhood, but most patients have a new mutation. Genetic analysis revealed that the FOP gene located on chromosome 4 and mutation in this gene causes an over expression of a bone morphogenetic protein (BMP4). This is characterized by proliferation of connective tissue in voluntary muscles, fascias, tendons and ligaments resulting clinically as painful swelling of the muscles and connective tissue. This swelling subsides, then after approximately 6 months or more, ossification starts at some sites at the mean age of 4-5 years. Eventually, heterotrophic bone formation interferes with normal movement of the patient and most of them are confined to a wheel chair by the third decade. Mortality is related to restrictive lung disease (inability to expand the chest). Congenital malformations which are characteristically observed in the great toes at birth in almost all cases of FOP are the diagnostic hallmark.A 2-year-old boy presented with left periscapular and upper back paraspinal tender masses. He was referred from pediatricians to our hospital (Imam Reza) and subsequently admitted. He was the third child of 37-year-old mother delivered by cesarean section with good Apgar score, normal birth weight (4100gr), head circumference (HC) of 35 cm and length of 53 cm. His parents were non consanguineous and the similar disease was not observed in the family. The mother was a general physician and the father was an engineer. The patient’s sister and brother were healthy. In the past medical history, atopic dermatitis and lacrimal duct obstruction were seen at infancy. Growth and development was normal in the past two years.Present disease began one year ago with painful swelling of the forehead after a mild trauma. Similar swelling in the arm and shoulders started later subsiding spontaneously. At this admission, he had paraspinal (upper thoracic and lower neck) and periscapular muscle swelling which was painful, tender and indurate, without redness and inflammation over the skin . The shoIn physical examination of the limbs, bilateral hallux valgus was observed .He was referred with the primary diagnosis of multiple hematomas due to coagulation disorders, in which routine blood tests were normal twice and only hypochrome microcytic anemia was present. Ultrasonography showed solid hypoechoic masses with the largest diameter of 85 mm and thickness of 26 mm adhered to the scapula and spine. Sonographic differential diagnoses were mesenchymal tumor, bone tumor and aneurysmal bone cyst.The first CT scan indicated soft tissue masses, 25 Hounsfield Unit in density without bone destruction or calcification propounding mesenchymal or vascular masses .In spite of typical clinical and imaging findings for the early stage of FOP such as the congenital malformation of bilateral hallux valgus as a hallmark in this step, the diagnosis was not made and an unnecessary biopsy was performed on the periscapular mass. It may have lead to catastrophic disability. The pathology report showed proliferation of fibro-connective tissue with large islets of compact cartilage cells and lacunars cells with blades and specula of bone and osteoblastic activation, compatible with fibrodysplasia ossificans progressiva. The patient was discharged with non steroid anti-inflammatory drugs. Almost one year later, extensive ossification was detected in foot and neck radiographies , chest CGay patin first described this entity (FOP) in 1648 as a case who “turned to wood”. Prevalence is approximately 1/2.000.000 and over 700 cases have been reported in the world literature to date.7]The radiological evidence of ectopic ossification is not usually detected until 6 months (sometimes up to one year), after the appearance of masses so the mean age at onset of ossification has been reported as 5 years. Bony bridge causes skeletal contracture  3]3]In our case, the onset of symptoms with the typical anatomic pattern occurred at one year of age and spread at the second year. Ossification in the radiologic exam and CT scan was detected. In addition, ossification of thoracic soft tissue causes scoliosis and restrictive pulmonary disease. As a result of this event, the patient will be predisposed to chest infection and consequently death in the third or fourth decade prevalently. 8]8]FOP should be diagnosed as early as possible based on history, clinical and imaging findings. The guide of diagnosis is bilateral anomaly of the great toes present from birth, reported almost in 100% 6]9] of[9] of6][ of[9] ofRoentgenogram aid demonstrates minor osseous dysmorphism such as hallux valgus, clinodactyly, microdactyly, phalangeal shortening, metacarpal and metatarsal shortening, shallow acetabulum, short widened femoral neck, medial cortex thickening of the tibia, long small vertebra, fusion and segmentation of the spine, increased incidence of exostosis and long bone metaphyseal widening.When the ectopic ossification occurs, in plane radiography, bony bridges running from the bones to the muscles spreading also along the fascial planes are seen 9]..9]. FUS of early lesions may show sonolucent soft tissue mass and then when ossification occurs, echogenic and shadowing mass is detected.2]2] In early disease before ossification, CT demonstrates fascia plane edema and swelling and mesenchymal mass like lesion in the muscle that moderately enhances with IV contrast. In late disease after ossification and calcification, these findings could be seen in the muscles, fascia and connective tissue, especially near the bones.3]3] Although the diagnosis of FOP is more difficult when ectopic ossification is not present, MRI finding of a preosseous lesion is sufficient for accurate diagnosis of FOP in early stage 9]. In T. In T9].Although other soft tissue tumors mimic this MRI finding, MRI appearance of spreaded masses along the fascial planes is the single feature that confirms the diagnosis. Late MRI shows low T1 and T2 signal intensity associated with fibrous or calcified tissue.Bone scintigraphy with TC-MDP demonstrates heterotrophic ossification in the early stage and helps in the assessment of the extent and progression of the disease. In our patient, plain radiography, US and CT scan were performed. US showed hypoechoic soft tissue masses near the left scapula and spine corresponding to mesenchymal tumor or bone tumor and CT scan showed soft tissue masses with 25 HU in density that adhered to the bone without bone distraction or obvious calcification, propounded mesenchymal or vascular massesIn the early phase, these patients may be misdiagnosed with malignant tumors especially sarcoma, rhabdomyosarcoma, aggressive fibromatosis, post traumatic myositis, lymphadenopathy, TB, scleroderma, dermatomyositis of childhood, RA, nodular fasciitis and Klippel-Feil syndrome. 5]8]9][8][9]5][[9][8][9][8][9]5]In the late phase, the differential diagnoses are osteosarcoma, progressive osseous heteroplasia (POH), Albright hereditary osteodystrophy (AHO), osteoma cutis, ankylosing spondylitis, Still's disease, calcinosis interstitialis ossificans, Weber-Christian disease, pseudohypoparathyroidism, hypervitaminosis D, traumatic myositis ossificans and multiple exostosis. 8]10][10]8][10[10]8][10FOP has many differential diagnoses. This disease may be misdiagnosed in the early stage most importantly and most commonly as sarcoma and other cancers, aggressive fibromatosis and vascular masses.In summary, in review of case series and case reports and articles of diagnostic errors, and our case, we conclude that FOP is often misdiagnosed and the correct diagnosis happens very late. Early diagnosis prevents catastrophic harmful diagnostic and treatment procedures. For early correct diagnosis we propose:1. Family physicians, pediatricians and radiologists should be aware of the early feature of FOP before development of heterotrophic ossification. The most important and best key is great toe malformation which must be noticed clinically and radiologically as an early pattern of involvement.2. Early clinical and radiological diagnosis must lead to an MRI study or molecular genetic study, which depends on the accessibility and cost. Both of them proved the diagnosis.