Teaching molecular genetics: chapter 4—positional cloning of genetic disorders

Positional cloning is the approach of choice for the identification of genetic mutations underlying the pathological development of diseases with simple Mendelian inheritance. It consists of different consecutive steps, starting with recruitment of patients and DNA collection, that are critical to the overall process. A genetic analysis of the enrolled patients and their families is performed, based on genetic recombination frequencies generated by meiotic cross-overs and on genome-wide molecular studies, to define a critical DNA region of interest. This analysis culminates in a statistical estimate of the probability that disease features may segregate in the families independently or in association with specific molecular markers located in known regions. In this latter case, a marker can be defined as being linked to the disease manifestations. The genetic markers define an interval that is a function of their recombination frequencies with the disease, in which the disease gene is localised. The identification and characterisation of chromosome abnormalities as translocations, deletions and duplications by classical cytogenetic methods or by the newly developed microarray-based comparative genomic hybridisation (array CGH) technique may define extensions and borders of the genomic regions involved. The step following the definition of a critical genomic region is the identification of candidate genes that is based on the analysis of available databases from genome browsers. Positional cloning culminates in the identification of the causative gene mutation, and the definition of its functional role in the pathogenesis of the disorder, by the use of cell-based or animal-based experiments. More often, positional cloning ends with the generation of mice with homologous mutations reproducing the human clinical phenotype. Altogether, positional cloning has represented a fundamental step in the research on genetic renal disorders, leading to the definition of several disease mechanisms and allowing a proper diagnostic approach to many conditions

Identification of Vascular and Hematopoietic Genes Downstream of etsrp by Deep Sequencing in Zebrafish

The transcription factor etsrp/Er71/Etv2 is a master control gene for vasculogenesis in all species studied to date. It is also required for hematopoiesis in zebrafish and mice. Several novel genes expressed in vasculature have been identified through transcriptional profiling of zebrafish embryos overexpressing etsrp by microarrays. Here we re-examined this transcriptional profile by Illumina RNA-sequencing technology, revealing a substantially increased number of candidate genes regulated by etsrp. Expression studies of 50 selected candidate genes from this dataset resulted in the identification of 39 new genes that are expressed in vascular cells. Regulation of these genes by etsrp was confirmed by their ectopic induction in etsrp overexpressing and decreased expression in etsrp deficient embryos. Our studies demonstrate the effectiveness of the RNA-sequencing technology to identify biologically relevant genes in zebrfish and produced a comprehensive profile of genes previously unexplored in vascular endothelial cell biology

The read-across hypothesis and environmental risk assessment of pharmaceuticals

This article is made available through the Brunel Open Access Publishing Fund. Copyright © 2013 American Chemical Society.Pharmaceuticals in the environment have received increased attention over the past decade, as they are ubiquitous in rivers and waterways. Concentrations are in sub-ng to low μg/L, well below acute toxic levels, but there are uncertainties regarding the effects of chronic exposures and there is a need to prioritise which pharmaceuticals may be of concern. The read-across hypothesis stipulates that a drug will have an effect in non-target organisms only if the molecular targets such as receptors and enzymes have been conserved, resulting in a (specific) pharmacological effect only if plasma concentrations are similar to human therapeutic concentrations. If this holds true for different classes of pharmaceuticals, it should be possible to predict the potential environmental impact from information obtained during the drug development process. This paper critically reviews the evidence for read-across, and finds that few studies include plasma concentrations and mode of action based effects. Thus, despite a large number of apparently relevant papers and a general acceptance of the hypothesis, there is an absence of documented evidence. There is a need for large-scale studies to generate robust data for testing the read-across hypothesis and developing predictive models, the only feasible approach to protecting the environment.BBSRC Industrial Partnership Award BB/ I00646X/1 and BBSRC Industrial CASE Partnership Studentship BB/I53257X/1 with AstraZeneca Safety Health and Environment Research Programme

Discovery and Characterization of Novel Vascular and Hematopoietic Genes Downstream of Etsrp in Zebrafish

The transcription factor Etsrp is required for vasculogenesis and primitive myelopoiesis in zebrafish. When ectopically expressed, etsrp is sufficient to induce the expression of many vascular and myeloid genes in zebrafish. The mammalian homolog of etsrp, ER71/Etv2, is also essential for vascular and hematopoietic development. To identify genes downstream of etsrp, gain-of-function experiments were performed for etsrp in zebrafish embryos followed by transcription profile analysis by microarray. Subsequent in vivo expression studies resulted in the identification of fourteen genes with blood and/or vascular expression, six of these being completely novel. Regulation of these genes by etsrp was confirmed by ectopic induction in etsrp overexpressing embryos and decreased expression in etsrp deficient embryos. Additional functional analysis of two newly discovered genes, hapln1b and sh3gl3, demonstrates their importance in embryonic vascular development. The results described here identify a group of genes downstream of etsrp likely to be critical for vascular and/or myeloid development

The ADMR Receptor Mediates the Effects of Adrenomedullin on Pancreatic Cancer Cells and on Cells of the Tumor Microenvironment

Adrenomedullin (AM) is highly expressed in pancreatic cancer and stimulates pancreatic cancer cells leading to increased tumor growth and metastasis. The current study examines the role of specific AM receptors on tumor and cells resembling the tumor microenvironment (human pancreatic stellate--HPSC, human umbilical vein-- HUVEC and mouse lung endothelial cells--MLEC).AM receptors ADMR and CRLR were present in HPSC, HUVEC and MLECs while PDAC cells possessed only ADMR receptors as assessed by RT-PCR and western blotting. All cell lines expressed and secreted AM as indicated by ELISA. The growth of each of the cell lines was stimulated by exogenous AM and inhibited by the antagonist AMA. AM also stimulated in vitro angiogenesis assessed by polygon formation of endothelial cell lines. SiRNA-mediated silencing of ADMR, but not CRLR, reduced basal growth of all cells examined and reduced polygon formation of endothelial cells in vitro. Orthotopic tumors developed with shADMR bearing cancer cells had dramatically reduced primary tumor volume (>90%) and lung and liver metastasis compared to shControl bearing cells. To validate ADMR as a potential therapeutic target, in vivo studies were conducted using neutral nanoliposomes to systemically deliver human siRNA to ADMR to silence human cancer cells and mouse siRNA to ADMR to silence mouse tumor stromal cells. Systemic silencing of both human and mouse ADMR had no obvious adverse effects but strongly reduced tumor development.ADMR mediates the stimulatory effects of AM on cancer cells and on endothelial and stellate cells within the tumor microenvironment. These data support the further development of ADMR as a useful target treatment of pancreatic cancer

A Novel and Critical Role for Oct4 as a Regulator of the Maternal-Embryonic Transition

Compared to the emerging embryonic stem cell (ESC) gene network, little is known about the dynamic gene network that directs reprogramming in the early embryo. We hypothesized that Oct4, an ESC pluripotency regulator that is also highly expressed at the 1- to 2-cell stages in embryos, may be a critical regulator of the earliest gene network in the embryo.Using antisense morpholino oligonucleotide (MO)-mediated gene knockdown, we show that Oct4 is required for development prior to the blastocyst stage. Specifically, Oct4 has a novel and critical role in regulating genes that encode transcriptional and post-transcriptional regulators as early as the 2-cell stage. Our data suggest that the key function of Oct4 may be to switch the developmental program from one that is predominantly regulated by post-transcriptional control to one that depends on the transcriptional network. Further, we propose to rank candidate genes quantitatively based on the inter-embryo variation in their differential expression in response to Oct4 knockdown. Of over 30 genes analyzed according to this proposed paradigm, Rest and Mta2, both of which have established pluripotency functions in ESCs, were found to be the most tightly regulated by Oct4 at the 2-cell stage.We show that the Oct4-regulated gene set at the 1- to 2-cell stages of early embryo development is large and distinct from its established network in ESCs. Further, our experimental approach can be applied to dissect the gene regulatory network of Oct4 and other pluripotency regulators to deconstruct the dynamic developmental program in the early embryo

A core human primary tumor angiogenesis signature identifies the endothelial orphan receptor ELTD1 as a key regulator of angiogenesis.

Limited clinical benefits derived from anti-VEGF therapy have driven the identification of new targets involved in tumor angiogenesis. Here, we report an integrative meta-analysis to define the transcriptional program underlying angiogenesis in human cancer. This approach identified ELTD1, an orphan G-protein-coupled receptor whose expression is induced by VEGF/bFGF and repressed by DLL4 signaling. Extensive analysis of multiple cancer types demonstrates significant upregulation of ELTD1 in tumor-associated endothelial cells, with a higher expression correlating with favorable prognosis. Importantly, ELTD1 silencing impairs endothelial sprouting and vessel formation in vitro and in vivo, drastically reducing tumor growth and greatly improving survival. Collectively, these results provide insight into the regulation of tumor angiogenesis and highlight ELTD1 as key player in blood vessel formation

Modulation of the β-Catenin Signaling Pathway by the Dishevelled-Associated Protein Hipk1

BACKGROUND:Wnts are evolutionarily conserved ligands that signal through beta-catenin-dependent and beta-catenin-independent pathways to regulate cell fate, proliferation, polarity, and movements during vertebrate development. Dishevelled (Dsh/Dvl) is a multi-domain scaffold protein required for virtually all known Wnt signaling activities, raising interest in the identification and functions of Dsh-associated proteins. METHODOLOGY:We conducted a yeast-2-hybrid screen using an N-terminal fragment of Dsh, resulting in isolation of the Xenopus laevis ortholog of Hipk1. Interaction between the Dsh and Hipk1 proteins was confirmed by co-immunoprecipitation assays and mass spectrometry, and further experiments suggest that Hipk1 also complexes with the transcription factor Tcf3. Supporting a nuclear function during X. laevis development, Myc-tagged Hipk1 localizes primarily to the nucleus in animal cap explants, and the endogenous transcript is strongly expressed during gastrula and neurula stages. Experimental manipulations of Hipk1 levels indicate that Hipk1 can repress Wnt/beta-catenin target gene activation, as demonstrated by beta-catenin reporter assays in human embryonic kidney cells and by indicators of dorsal specification in X. laevis embryos at the late blastula stage. In addition, a subset of Wnt-responsive genes subsequently requires Hipk1 for activation in the involuting mesoderm during gastrulation. Moreover, either over-expression or knock-down of Hipk1 leads to perturbed convergent extension cell movements involved in both gastrulation and neural tube closure. CONCLUSIONS:These results suggest that Hipk1 contributes in a complex fashion to Dsh-dependent signaling activities during early vertebrate development. This includes regulating the transcription of Wnt/beta-catenin target genes in the nucleus, possibly in both repressive and activating ways under changing developmental contexts. This regulation is required to modulate gene expression and cell movements that are essential for gastrulation

The EYA Tyrosine Phosphatase Activity Is Pro-Angiogenic and Is Inhibited by Benzbromarone

Eyes Absents (EYA) are multifunctional proteins best known for their role in organogenesis. There is accumulating evidence that overexpression of EYAs in breast and ovarian cancers, and in malignant peripheral nerve sheath tumors, correlates with tumor growth and increased metastasis. The EYA protein is both a transcriptional activator and a tyrosine phosphatase, and the tyrosine phosphatase activity promotes single cell motility of mammary epithelial cells. Since EYAs are expressed in vascular endothelial cells and cell motility is a critical feature of angiogenesis we investigated the role of EYAs in this process. Using RNA interference techniques we show that EYA3 depletion in human umbilical vein endothelial cells inhibits transwell migration as well as Matrigel-induced tube formation. To specifically query the role of the EYA tyrosine phosphatase activity we employed a chemical biology approach. Through an experimental screen the uricosuric agents Benzbromarone and Benzarone were found to be potent EYA inhibitors, and Benzarone in particular exhibited selectivity towards EYA versus a representative classical protein tyrosine phosphatase, PTP1B. These compounds inhibit the motility of mammary epithelial cells over-expressing EYA2 as well as the motility of endothelial cells. Furthermore, they attenuate tubulogenesis in matrigel and sprouting angiogenesis in the ex vivo aortic ring assay in a dose-dependent fashion. The anti-angiogenic effect of the inhibitors was also demonstrated in vivo, as treatment of zebrafish embryos led to significant and dose-dependent defects in the developing vasculature. Taken together our results demonstrate that the EYA tyrosine phosphatase activity is pro-angiogenic and that Benzbromarone and Benzarone are attractive candidates for repurposing as drugs for the treatment of cancer metastasis, tumor angiogenesis, and vasculopathies