Mutations in the colony stimulating factor 1 receptor (CSF1R) gene cause hereditary diffuse leukoencephalopathy with spheroids.

Hereditary diffuse leukoencephalopathy with spheroids (HDLS) is an autosomal-dominant central nervous system white-matter disease with variable clinical presentations, including personality and behavioral changes, dementia, depression, parkinsonism, seizures and other phenotypes. We combined genome-wide linkage analysis with exome sequencing and identified 14 different mutations affecting the tyrosine kinase domain of the colony stimulating factor 1 receptor (encoded by CSF1R) in 14 families with HDLS. In one kindred, we confirmed the de novo occurrence of the mutation. Follow-up sequencing identified an additional CSF1R mutation in an individual diagnosed with corticobasal syndrome. In vitro, CSF-1 stimulation resulted in rapid autophosphorylation of selected tyrosine residues in the kinase domain of wild-type but not mutant CSF1R, suggesting that HDLS may result from partial loss of CSF1R function. As CSF1R is a crucial mediator of microglial proliferation and differentiation in the brain, our findings suggest an important role for microglial dysfunction in HDLS pathogenesis.

Translocation of the yeast dolichol-phosphate-mannose synthase into microsomal membranes.

Dolichol-phosphate-mannose synthase catalyzes the formation of Dolichol-phosphate-mannose from Dolichol-phosphate and GDP-mannose. Analysis of the primary amino acid sequence of the yeast enzyme predicts a luminal orientation of the enzyme in the endoplasmic reticulum. We analysed the translocation of the Dolichol-phosphate-mannose synthase into dog pancreatic microsomal membranes: resistance to proteolytic attack provides evidence of its luminal orientation and asks for a reevaluation of the topology of the reaction.

Superfamily of steroid nuclear receptors: positive and negative regulators of gene expression.

The nuclear hormone receptor superfamily is characterized by an impressive functional diversity of its members despite a remarkable overall structural unity. A variety of ligands bind specifically to them and these receptors control gene networks that have profound effects on growth, development, and homeostasis. The ligand-receptor complexes recognize transcriptional enhancer DNA sequences, the hormone response elements, resulting in induction or repression of gene activity. The similarity between all these hormone response enhancer elements, as well as between the receptors themselves, indicates a conserved general strategy for the hormonal control of transcription by steroids. The activated receptors bind to responsive promoters and most likely mediate the assembly of stage- and tissue-specific transcription factor complexes that stimulate or inhibit gene expression.

Recurrent loss of heterozygosity in 1p36 associated with TNFRSF14 mutations in IRF4 translocation negative pediatric follicular lymphomas.

Pediatric follicular lymphoma is a rare disease that differs genetically and clinically from its adult counterpart. With the exception of pediatric follicular lymphoma with IRF4-translocation, the genetic events associated with these lymphomas have not yet been defined. We applied array-comparative genomic hybridization and molecular inversion probe assay analyses to formalin-fixed paraffin-embedded tissues from 18 patients aged 18 years and under with IRF4 translocation negative follicular lymphoma. All evaluable cases lacked t(14;18). Only 6 of 16 evaluable cases displayed chromosomal imbalances with gains or amplifications of 6pter-p24.3 (including IRF4) and deletion and copy number neutral-loss of heterozygosity in 1p36 (including TNFRSF14) being most frequent. Sequencing of TNFRSF14 located in the minimal region of loss in 1p36.32 showed nine mutations in 7 cases from our series. Two subsets of pediatric follicular lymphoma were delineated according to the presence of molecular alterations, one with genomic aberrations associated with higher grade and/or diffuse large B-cell lymphoma component and more widespread disease, and another one lacking genetic alterations associated with more limited disease.

Low gene copy number shows that arbuscular mycorrhizal fungi inherit genetically different nuclei.

Arbuscular mycorrhizal fungi (AMF) are ancient asexually reproducing organisms that form symbioses with the majority of plant species, improving plant nutrition and promoting plant diversity. Little is known about the evolution or organization of the genomes of any eukaryotic symbiont or ancient asexual organism. Direct evidence shows that one AMF species is heterokaryotic; that is, containing populations of genetically different nuclei. It has been suggested, however, that the genetic variation passed from generation to generation in AMF is simply due to multiple chromosome sets (that is, high ploidy). Here we show that previously documented genetic variation in Pol-like sequences, which are passed from generation to generation, cannot be due to either high ploidy or repeated gene duplications. Our results provide the clearest evidence so far for substantial genetic differences among nuclei in AMF. We also show that even AMF with a very large nuclear DNA content are haploid. An underlying principle of evolutionary theory is that an individual passes on one or half of its genome to each of its progeny. The coexistence of a population of many genomes in AMF and their transfer to subsequent generations, therefore, has far-reaching consequences for understanding genome evolution.

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.

EuroDia: a beta-cell gene expression resource.

Type 2 diabetes mellitus (T2DM) is a major disease affecting nearly 280 million people worldwide. Whilst the pathophysiological mechanisms leading to disease are poorly understood, dysfunction of the insulin-producing pancreatic beta-cells is key event for disease development. Monitoring the gene expression profiles of pancreatic beta-cells under several genetic or chemical perturbations has shed light on genes and pathways involved in T2DM. The EuroDia database has been established to build a unique collection of gene expression measurements performed on beta-cells of three organisms, namely human, mouse and rat. The Gene Expression Data Analysis Interface (GEDAI) has been developed to support this database. The quality of each dataset is assessed by a series of quality control procedures to detect putative hybridization outliers. The system integrates a web interface to several standard analysis functions from R/Bioconductor to identify differentially expressed genes and pathways. It also allows the combination of multiple experiments performed on different array platforms of the same technology. The design of this system enables each user to rapidly design a custom analysis pipeline and thus produce their own list of genes and pathways. Raw and normalized data can be downloaded for each experiment. The flexible engine of this database (GEDAI) is currently used to handle gene expression data from several laboratory-run projects dealing with different organisms and platforms. Database URL: http://eurodia.vital-it.ch.

CYP3A activity measured by the midazolam test is not related to 3435 C >T polymorphism in the multiple drug resistance transporter gene.

A recent study with 69 Japanese liver transplants treated with tacrolimus found that the MDR13435 C >T polymorphism, but not the MDR12677 G >T polymorphism, was associated with differences in the intestinal expression level of CYP3A4 mRNA. In the present study, over 6 h, we measured the kinetics of a 75 microg oral dose of midazolam, a CYP3A substrate, in 21 healthy subjects genotyped for the MDR13435 C >T and 2677 G >T polymorphism. No statistically significant differences were found in the calculated pharmacokinetic parameters between the three 3435 C >T genotypes (TT, CT and CC group, respectively: Cmax (mean +/- SD: 0.30 +/- 0.08 ng/ml, 0.31 +/- 0.09 ng/ml and 0.31 +/- 0.11 ng/ml; Apparent clearance: 122 +/- 29 l/h, 156 +/- 92 l/h and 111 +/- 35 l/h; t1/2: 1.9 +/- 1.1 h, 1.6 +/- 0.90 h and 1.7 +/- 0.7 h). In addition, the 30-min 1'OH midazolam to midazolam ratio, a marker of CYP3A activity, determined in 74 HIV-positive patients before the introduction of antiretroviral treatment, was not significantly different between the three 3435 C >T genotypes (mean ratio +/- SD: 3.65 +/- 2.24, 4.22 +/- 3.49 and 4.24 +/- 2.03, in the TT, CT and CC groups, respectively). Similarly, no association was found between the MDR12677 G >T polymorphism and CYP3A activity in the healthy subjects or in the HIV-positive patients. The existence of a strong association between the activity of CYP3A and MDR13435 C >T and 2677 G >T polymorphisms appears unlikely, at least in Caucasian populations and/or in the absence of specific environmental factors.

Development of Ewing's sarcoma from primary bone marrow-derived mesenchymal stem cells

SUMMARY : Ewing's sarcoma is a member of Ewing's family tumors (ESPY) and the second most common solid bone and soft tissue malignancy of children and young adults. It is associated in 85% of cases with the t(11;22)(q24:q12) chromosomal translocation that generates fusion of the 5' segment of the EWSR1 gene with the 3' segment of the ETS family gene FLI-1. The EWSR1-FLI-1 fusion protein behaves as an aberrant transcriptional activator and is believed to contribute to ESFT development. However, EWSR1-FLI-1 induces growth arrest and apoptosis in normal fibroblasts, and primary cells that are pemissive for its putative oncogenic properties have not been discovered, hampering basic understanding of ESFT biology. Here, we show that EWSR1-FLI-1 alone can transform mouse primary bone marrow-derived mesenchymal progenitor cells and generate tumors that display hallmarks of Ewing's sarcoma, including a small round cell phenotype, expression of ESFT-associated markers, insulin like growth factor-I dependence, and induction or repression of numerous EWSR1-FLI-1 target genes. Consistent with this finding, we tested the possibility that human mesenchymal stem cells (hMSC) might also provide a permissive cellular environment for EWSR1-FLI-1, and could represent the first adequate primary human cellular background for the oncogenic properties of the fusion protein. Indeed, expression of EWSR1-FLI-1 in human mesenchymal stem cells (hMSC) was not only stably maintained without inhibiting proliferation, but induced a gene expression profile bearing striking similarity to that of ESFT, including genes that are among the highest ESFT discriminators. Expression of EWSR1-FLI-1 in hMSCs may recapitulate the initial steps of Ewing's sarcoma development, allowing identification of genes that play an important role early in its pathogenesis. Among relevant candidate transcripts induced by EWSR1-FL/-1 in hMSC we found the polycomb group gene EZH2 which we show to play a critical role in Ewing's sarcoma growth. These observations provide the first identification of candidate primary cells from which ESFTs originate and suggest that EWSR1-FLI-1 expression may constitute the initiating event in ESFT pathogenesis. Le sarcome d' Ewing est un membre de la famille des tumeurs Ewing (ESFT) et représente la deuxième tumeur maligne solide de l'os et des tissus mous chez les enfants et les jeunes adultes. Cette tumeur est associée dans 85% des cas avec la translocation chromosomique t(11;22)(g24:g12), qui génère la fusion entre le segment 5' du gène EWSR1 avec le segment 3' du gène FLI-1, appartenant à la famille des facteurs de transcription ETS. La protéine de fusion EWSR1-FLI-1 qui en dérive joue le rSle d'un facteur de transcription aberrant, et est supposée contribuer de manière décisive au processus de développement des ESFTs. Néanmoins, l'expression de EWSR1-FLI-1 dans des fibroblastes normaux induit un arrêt de croissance et leur apoptose, et les cellules primaires permissives pour les propriétés oncogéniques attribuées à la translocation n'ont pas encore été identifiées, empêchant la compréhension de la biologie de base du sarcome d'Ewing. Dans ce travail on montre que l'expression de EWSR1-FLI-1 uniquement est capable de transformer des cellules souches mésenchymateuses dérivées de la moelle osseuse de la souris, pour générer des tumeurs qui présentent les caractéristiques du sarcome d' Ewing humain, et notamment une morphologie de petites cellules bleues et rondes, l'expression de marqueurs associés aux ESFTs, une dépendance du facteur de croissance IGF-1, et l'induction ou la répression de nombreux gènes cibles connus de EWSR1-FLI-1. Sur la base de ces observations, on a testé la possibilité que les cellules souches mésenchymateuses humaines (hMSCs) puissent aussi fournir un environnement cellulaire permissif pour EWSR1-FLI-1 ; et représenter le premier background cellulaire humain adéquat pour la manifestation du pouvoir oncogénique de la protéine de fusion. En effet, l'expression de EWSR1-FLI-1 dans des cellules souches mésenchymateuses humaines s'est révélée non seulement maintenue, mais elle a induit un profil d'expression génétique étonnamment similaire à celui des ESFTs humains, incluant les gènes qui ont été rapportés comme étant les plus discriminatifs pour ces tumeurs. L'expression de EWSR1-FLI-1 dans les hMSCs pourrait récapituler les étapes initiales du développement du sarcome d' Ewing, et de ce fait consentir à identifier les gènes qui jouent un rôle crucial dans sa pathogenèse précoce. Parmi les transcrits relevant indults par EWSR1-FL/-9 dans les hMSCs nous avons découvert le gène du groupe des polycomb EZH2, que nous avons par la suite démontré jouer un rôle essentiel dans la croissance du sarcome de Ewing. Ces observations apportent pour la première fois l'identification d'une cellule primaire candidate pour représenter la cellule d'origine des ESFTs, et en même temps suggèrent que l'expression de EWSR1-FLI-1 peut constituer l'événement initial dans la pathogenèse du sarcome d' Ewing.

Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor betaPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.

Arenavirus Nucleoprotein Targets Interferon Regulatory Factor-Activating Kinase IKKε.

Arenaviruses perturb innate antiviral defense by blocking induction of type I interferon (IFN) production. Accordingly, the arenavirus nucleoprotein (NP) was shown to block activation and nuclear translocation of interferon regulatory factor 3 (IRF3) in response to virus infection. Here, we sought to identify cellular factors involved in innate antiviral signaling targeted by arenavirus NP. Consistent with previous studies, infection with the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) prevented phosphorylation of IRF3 in response to infection with Sendai virus, a strong inducer of the retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS) pathway of innate antiviral signaling. Using a combination of coimmunoprecipitation and confocal microscopy, we found that LCMV NP associates with the IκB kinase (IKK)-related kinase IKKε but that, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKKε interaction was highly conserved among arenaviruses from different clades. In LCMV-infected cells, IKKε colocalized with NP but not with MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKKε (IKBKE) and blocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our data identify IKKε as a novel target of arenavirus NP. Engagement of NP seems to sequester IKKε in an inactive complex. Considering the important functions of IKKε in innate antiviral immunity and other cellular processes, the NP-IKKε interaction likely plays a crucial role in arenavirus-host interaction.

Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.

Characterization of pip5k3 fleck corneal dystrophy-linked gene in zebrafish.

PIKfyve is a kinase encoded by pip5k3 involved in phosphatidylinositols (PdtIns) pathways. These lipids building cell membranes have structural functions and are involved in complex intracellular regulations. Mutations in human PIP5K3 are associated with François-Neetens mouchetée fleck corneal dystrophy [Li, S., Tiab, L., Jiao, X., Munier, F.L., Zografos, L., Frueh, B.E., Sergeev, Y., Smith, J., Rubin, B., Meallet, M.A., Forster, R.K., Hejtmancik, J.F., Schorderet, D.F., 2005. Mutations in PIP5K3 are associated with François-Neetens mouchetee fleck corneal dystrophy. Am. J. Hum. Genet. 77, 54-63]. We cloned the zebrafish pip5k3 and report its molecular characterization and expression pattern in adult fish as well as during development. The zebrafish PIKfyve was 70% similar to the human homologue. The gene encompassed 42 exons and presented four alternatively spliced variants. It had a widespread expression in the adult organs and was localized in specific cell types in the eye as the cornea, lens, ganglion cell layer, inner nuclear layer and outer limiting membrane. Pip5k3 transcripts were detected in early cleavage stage embryos. Then it was uniformly expressed at 10 somites, 18 somites and 24 hpf. Its expression was then restricted to the head region at 48 hpf, 72 hpf and 5 dpf and partial expression was found in somites at 72 hpf and 5 dpf. In situ on eye sections at 3 dpf showed a staining mainly in lens, outer limiting membrane, inner nuclear layer and ganglion cell layer. A similar expression pattern was found in the eye at 5 dpf. A temporal regulation of the spliced variants was observed at 1, 3 and 5 dpf and they were also found in the adult eye.