A comprehensive analysis of gene expression profiles in distal parts of the mouse renal tubule.

The distal parts of the renal tubule play a critical role in maintaining homeostasis of extracellular fluids. In this review, we present an in-depth analysis of microarray-based gene expression profiles available for microdissected mouse distal nephron segments, i.e., the distal convoluted tubule (DCT) and the connecting tubule (CNT), and for the cortical portion of the collecting duct (CCD; Zuber et al., Proc Natl Acad Sci USA 106:16523-16528, 2009). Classification of expressed transcripts in 14 major functional gene categories demonstrated that all principal proteins involved in maintaining the salt and water balance are represented by highly abundant transcripts. However, a significant number of transcripts belonging, for instance, to categories of G-protein-coupled receptors or serine/threonine kinases exhibit high expression levels but remain unassigned to a specific renal function. We also established a list of genes differentially expressed between the DCT/CNT and the CCD. This list is enriched by genes related to segment-specific transport functions and by transcription factors directing the development of the distal nephron or collecting ducts. Collectively, this in silico analysis provides comprehensive information about relative abundance and tissue specificity of the DCT/CNT and the CCD expressed transcripts and identifies new candidate genes for renal homeostasis.

New gene functions in megakaryopoiesis and platelet formation.

Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function.

Interferon-Induced Gene Expression Is a Stronger Predictor of Treatment Response Than IL28B Genotype in Patients With Hepatitis C.

BACKGROUND & AIMS: The host immune response during the chronic phase of hepatitis C virus infection varies among individuals; some patients have a no interferon (IFN) response in the liver, whereas others have full activation IFN-stimulated genes (ISGs). Preactivation of this endogenous IFN system is associated with nonresponse to pegylated IFN-α (pegIFN-α) and ribavirin. Genome-wide association studies have associated allelic variants near the IL28B (IFNλ3) gene with treatment response. We investigated whether IL28B genotype determines the constitutive expression of ISGs in the liver and compared the abilities of ISG levels and IL28B genotype to predict treatment outcome. METHODS: We genotyped 109 patients with chronic hepatitis C for IL28B allelic variants and quantified the hepatic expression of ISGs and of IL28B. Decision tree ensembles, in the form of a random forest classifier, were used to calculate the relative predictive power of these different variables in a multivariate analysis. RESULTS: The minor IL28B allele was significantly associated with increased expression of ISG. However, stratification of the patients according to treatment response revealed increased ISG expression in nonresponders, irrespective of IL28B genotype. Multivariate analysis of ISG expression, IL28B genotype, and several other factors associated with response to therapy identified ISG expression as the best predictor of treatment response. CONCLUSIONS: IL28B genotype and hepatic expression of ISGs are independent predictors of response to treatment with pegIFN-α and ribavirin in patients with chronic hepatitis C. The most accurate prediction of response was obtained with a 4-gene classifier comprising IFI27, ISG15, RSAD2, and HTATIP2.

A peptide encoded by the human MAGE3 gene and presented by HLA-B44 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE3.

The human MAGE3 gene is expressed in a significant proportion of tumors of various histological types, but is silent in normal adult tissues other than testis and placenta. Antigens encoded by MAGE3 may therefore be useful targets for specific antitumor immunization. Two antigenic peptides encoded by the MAGE3 gene have been reported previously. One is presented to cytolytic T lymphocytes (CTL) by HLA-A1, the other by HLA-A2 molecules. Here we show that MAGE3 also codes for a peptide that is presented to CTL by HLA-B44. MAGE3 peptides containing the HLA-B44 peptide binding motif were synthesized. Peptide MEVDPIGHLY, which showed the strongest binding to HLA-B44, was used to stimulate blood T lymphocytes from normal HLA-B44 donors. CTL clones were obtained that recognized not only HLA-B44 cells sensitized with the peptide, but also HLA-B44 tumor cell lines expressing MAGE3. The proportion of metastatic melanomas expressing the MAGE3/HLA-B44 antigen should amount to approximately 17% in the Caucasian population, since 24% of individuals carry the HLA-B44 allele and 76% of these tumors express MAGE3.

Cross-species and cross-platform gene expression studies with the Bioconductor-compliant R package 'annotationTools'.

Background: The variety of DNA microarray formats and datasets presently available offers an unprecedented opportunity to perform insightful comparisons of heterogeneous data. Cross-species studies, in particular, have the power of identifying conserved, functionally important molecular processes. Validation of discoveries can now often be performed in readily available public data which frequently requires cross-platform studies.Cross-platform and cross-species analyses require matching probes on different microarray formats. This can be achieved using the information in microarray annotations and additional molecular biology databases, such as orthology databases. Although annotations and other biological information are stored using modern database models ( e. g. relational), they are very often distributed and shared as tables in text files, i.e. flat file databases. This common flat database format thus provides a simple and robust solution to flexibly integrate various sources of information and a basis for the combined analysis of heterogeneous gene expression profiles.Results: We provide annotationTools, a Bioconductor-compliant R package to annotate microarray experiments and integrate heterogeneous gene expression profiles using annotation and other molecular biology information available as flat file databases. First, annotationTools contains a specialized set of functions for mining this widely used database format in a systematic manner. It thus offers a straightforward solution for annotating microarray experiments. Second, building on these basic functions and relying on the combination of information from several databases, it provides tools to easily perform cross-species analyses of gene expression data.Here, we present two example applications of annotationTools that are of direct relevance for the analysis of heterogeneous gene expression profiles, namely a cross-platform mapping of probes and a cross-species mapping of orthologous probes using different orthology databases. We also show how to perform an explorative comparison of disease-related transcriptional changes in human patients and in a genetic mouse model.Conclusion: The R package annotationTools provides a simple solution to handle microarray annotation and orthology tables, as well as other flat molecular biology databases. Thereby, it allows easy integration and analysis of heterogeneous microarray experiments across different technological platforms or species.

Lentiviral-mediated gene transfer of siRNAs for the treatment of Huntington's disease.

This chapter describes the potential use of viral-mediated gene transfer in the central nervous system for the silencing of gene expression using RNA interference in the context of Huntington's disease (HD). Protocols provided here describe the design of small interfering RNAs, their encoding in lentiviral vectors (LVs) and viral production, as well as procedures for their stereotaxic injection in the rodent brain.

Adaptive divergence of ancient gene duplicates in the avian MHC class II beta.

Gene duplication and neofunctionalization are known to be important processes in the evolution of phenotypic complexity. They account for important evolutionary novelties that confer ecological adaptation, such as the major histocompatibility complex (MHC), a multigene family crucial to the vertebrate immune system. In birds, two MHC class II β (MHCIIβ) exon 3 lineages have been recently characterized, and two hypotheses for the evolutionary history of MHCIIβ lineages were proposed. These lineages could have arisen either by 1) an ancient duplication and subsequent divergence of one paralog or by 2) recent parallel duplications followed by functional convergence. Here, we compiled a data set consisting of 63 MHCIIβ exon 3 sequences from six avian orders to distinguish between these hypotheses and to understand the role of selection in the divergent evolution of the two avian MHCIIβ lineages. Based on phylogenetic reconstructions and simulations, we show that a unique duplication event preceding the major avian radiations gave rise to two ancestral MHCIIβ lineages that were each likely lost once later during avian evolution. Maximum likelihood estimation shows that following the ancestral duplication, positive selection drove a radical shift from basic to acidic amino acid composition of a protein domain facing the α-chain in the MHCII α β-heterodimer. Structural analyses of the MHCII α β-heterodimer highlight that three of these residues are potentially involved in direct interactions with the α-chain, suggesting that the shift following duplication may have been accompanied by coevolution of the interacting α- and β-chains. These results provide new insights into the long-term evolutionary relationships among avian MHC genes and open interesting perspectives for comparative and population genomic studies of avian MHC evolution.

Infrequent expression of the MAGE gene family in uveal melanomas.

It has previously been reported that MAGE-1, -2, -3 and -4 genes are expressed in human cancers including cutaneous melanoma. MAGE-1 and MAGE-3 represent targets for specific immunotherapy because they encode peptide antigens which are recognised by cytotoxic T lymphocytes (CTL) when presented by HLA class I molecules, and pilot clinical trials with these peptides are currently in progress. It is likely that other members of the MAGE gene family may also encode antigens recognised by CTL. Uveal melanomas, like cutaneous melanomas, arise from melanocytes that are derived from the neural crest. To determine if uveal melanoma patients would be suitable for MAGE-peptide immunotherapy, the expression of MAGE-1, -2, -3 and -4 genes was assessed by reverse transcription followed by polymerase chain reaction (RT-PCR) amplification and ethidium bromide staining. Expression of MAGE genes was not detected in any of 27 primary tumours. Either MAGE-1 or MAGE-4 was expressed in only 2 of 26 metastatic samples, but expression of MAGE-2 or -3 was not detected. Our data suggest that, unlike cutaneous melanomas, uveal melanomas may not be suitable candidates for MAGE-peptide immunotherapy.

Ins1 (Cre) knock-in mice for beta cell-specific gene recombination.

AIMS/HYPOTHESIS: Pancreatic beta cells play a central role in the control of glucose homeostasis by secreting insulin to stimulate glucose uptake by peripheral tissues. Understanding the molecular mechanisms that control beta cell function and plasticity has critical implications for the pathophysiology and therapy of major forms of diabetes. Selective gene inactivation in pancreatic beta cells, using the Cre-lox system, is a powerful approach to assess the role of particular genes in beta cells and their impact on whole body glucose homeostasis. Several Cre recombinase (Cre) deleter mice have been established to allow inactivation of genes in beta cells, but many show non-specific recombination in other cell types, often in the brain. METHODS: We describe the generation of Ins1 (Cre) and Ins1 (CreERT2) mice in which the Cre or Cre-oestrogen receptor fusion protein (CreERT2) recombinases have been introduced at the initiation codon of the Ins1 gene. RESULTS: We show that Ins1 (Cre) mice induce efficient and selective recombination of floxed genes in beta cells from the time of birth, with no recombination in the central nervous system. These mice have normal body weight and glucose homeostasis. Furthermore, we show that tamoxifen treatment of adult Ins1 (CreERT2) mice crossed with Rosa26-tdTomato mice induces efficient recombination in beta cells. CONCLUSIONS/INTERPRETATION: These two strains of deleter mice are useful new resources to investigate the molecular physiology of pancreatic beta cells.

Adenovirus-mediated gene transfer into selected liver segments using a vascular exclusion technique.

Adenovirus-mediated gene therapy is hampered by severe virus-related toxicity, especially to the liver. The aim of the present study was to test the ability of a vascular exclusion technique to achieve transgene expression within selected liver segments, thus minimizing both viral and transgene product toxicity to the liver. An E1-E3-deleted replication-deficient adenovirus expressing a green fluorescent protein (GFP) reporter gene was injected into the portal vein of BDIX rats, with simultaneous clamping of the portal vein tributaries to liver segments II, III, IV, V, and VIII. GFP expression and inflammatory infiltrate were measured in the different segments of the liver and compared with those of the livers of animals receiving the viral vector in the portal vein without clamping. The GFP expression was significantly higher in the selectively perfused segments of the liver as compared with the non-perfused segments (p < 0.0001) and with the livers of animals that received the vector in the portal vein without clamping (p < 0.0001). Accordingly, the inflammatory infiltrate was more intense in the selectively perfused liver segments as compared with all other groups (p < 0.0001). Fluorescence was absent in lungs and kidneys and minimal in spleen. The clinical usefulness of adenovirus-mediated gene transfer to the liver largely depends on the reduction of its liver toxicity. Clamping of selected portal vein branches during injection allows for delivery of genes of interest to targeted liver segments. Transgene expression confined to selected liver segments may be useful in the treatment of focal liver diseases, including metastases.

Genomic organization and gene expression in a chromosomal region of Leishmania major.

Little is known about the relation between the genome organization and gene expression in Leishmania. Bioinformatic analysis can be used to predict genes and find homologies with known proteins. A model was proposed, in which genes are organized into large clusters and transcribed from only one strand, in the form of large polycistronic primary transcripts. To verify the validity of this model, we studied gene expression at the transcriptional, post-transcriptional and translational levels in a unique locus of 34kb located on chr27 and represented by cosmid L979. Sequence analysis revealed 115 ORFs on either DNA strand. Using computer programs developed for Leishmania genes, only nine of these ORFs, localized on the same strand, were predicted to code for proteins, some of which show homologies with known proteins. Additionally, one pseudogene, was identified. We verified the biological relevance of these predictions. mRNAs from nine predicted genes and proteins from seven were detected. Nuclear run-on analyses confirmed that the top strand is transcribed by RNA polymerase II and suggested that there is no polymerase entry site. Low levels of transcription were detected in regions of the bottom strand and stable transcripts were identified for four ORFs on this strand not predicted to be protein-coding. In conclusion, the transcriptional organization of the Leishmania genome is complex, raising the possibility that computer predictions may not be comprehensive.

Focal dermal hypoplasia (goltz-gorlin syndrome): A new case with a novel variant in the PORCN gene (c.1250T>C:p.F417S) and unusual spinal anomaly.

Focal dermal hypoplasia (FDH; Goltz-Gorlin syndrome; OMIM 305600) is a disorder that features involvement of the skin, skeletal system, and eyes. It is caused by loss-of-function mutations in the PORCN gene. We report a young girl with FDH, microphthalmos associated with colobomatous orbital cyst, dural ectasia and cystic malformation of the spinal cord, and a de novo variant in PORCN. This association has not been previously reported, and based on these observations the phenotypic spectrum of FDH might be broader than previously appreciated. It would be prudent to alter the suggested surveillance for this rare disorder. © 2013 Wiley Periodicals, Inc.

Impact of salt on cardiac differential gene expression and coronary lesion in normotensive mineralocorticoid-treated mice.

We previously reported that excess of deoxycorticosterone-acetate (DOCA)/salt-induced cardiac hypertrophy in the absence of hypertension in one-renin gene mice. This model allows us to study molecular mechanisms of high-salt intake in the development of cardiovascular remodeling, independently of blood pressure in a high mineralocorticoid state. In this study, we compared the effect of 5-wk low- and high-salt intake on cardiovascular remodeling and cardiac differential gene expression in mice receiving the same amount of DOCA. Differential gene and protein expression was measured by high-density cDNA microarray assays, real-time PCR and Western blot analysis in DOCA-high salt (HS) vs. DOCA-low salt (LS) mice. DOCA-HS mice developed cardiac hypertrophy, coronary perivascular fibrosis, and left ventricular dysfunction. Differential gene and protein expression demonstrated that high-salt intake upregulated a subset of genes encoding for proteins involved in inflammation and extracellular matrix remodeling (e.g., Col3a1, Col1a2, Hmox1, and Lcn2). A major subset of downregulated genes encoded for transcription factors, including myeloid differentiation primary response (MyD) genes. Our data provide some evidence that vascular remodeling, fibrosis, and inflammation are important consequences of a high-salt intake in DOCA mice. Our study suggests that among the different pathogenic factors of cardiac and vascular remodeling, such as hypertension and mineralocorticoid excess and sodium intake, the latter is critical for the development of the profibrotic and proinflammatory phenotype observed in the heart of normotensive DOCA-treated mice.

Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition.

BACKGROUND: Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. RESULTS: The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window. CONCLUSION: Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking.