Deficiency in origin licensing proteins impairs cilia formation: implications for the aetiology of meier-gorlin syndrome

Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS), a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA-mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency

Widespread Contribution of Gdf7 Lineage to Cerebellar Cell Types and Implications for Hedgehog-Driven Medulloblastoma Formation

The roof plate is a specialized embryonic midline tissue of the central nervous system that functions as a signaling center regulating dorsal neural patterning. In the developing hindbrain, roof plate cells express Gdf7 and previous genetic fate mapping studies showed that these cells contribute mostly to non-neural choroid plexus epithelium. We demonstrate here that constitutive activation of the Sonic hedgehog signaling pathway in the Gdf7 lineage invariably leads to medulloblastoma. Lineage tracing analysis reveals that Gdf7-lineage cells not only are a source of choroid plexus epithelial cells, but are also present in the cerebellar rhombic lip and contribute to a subset of cerebellar granule neuron precursors, the presumed cell-of-origin for Sonic hedgehog-driven medulloblastoma. We further show that Gdf7-lineage cells also contribute to multiple neuronal and glial cell types in the cerebellum, including glutamatergic granule neurons, unipolar brush cells, Purkinje neurons, GABAergic interneurons, Bergmann glial cells, and white matter astrocytes. These findings establish hindbrain roof plate as a novel source of diverse neural cell types in the cerebellum that is also susceptible to oncogenic transformation by deregulated Sonic hedgehog signaling

Strong signature of natural selection within an FHIT intron implicated in prostate cancer risk

Previously, a candidate gene linkage approach on brother pairs affected with prostate cancer identified a locus of prostate cancer susceptibility at D3S1234 within the fragile histidine triad gene (FHIT), a tumor suppressor that induces apoptosis. Subsequent association tests on 16 SNPs spanning approximately 381 kb surrounding D3S1234 in Americans of European descent revealed significant evidence of association for a single SNP within intron 5 of FHIT. In the current study, resequencing and genotyping within a 28.5 kb region surrounding this SNP further delineated the association with prostate cancer risk to a 15 kb region. Multiple SNPs in sequences under evolutionary constraint within intron 5 of FHIT defined several related haplotypes with an increased risk of prostate cancer in European-Americans. Strong associations were detected for a risk haplotype defined by SNPs 138543, 142413, and 152494 in all cases (Pearson's χ2 = 12.34, df 1, P = 0.00045) and for the homozygous risk haplotype defined by SNPs 144716, 142413, and 148444 in cases that shared 2 alleles identical by descent with their affected brothers (Pearson's χ2 = 11.50, df 1, P = 0.00070). In addition to highly conserved sequences encompassing SNPs 148444 and 152413, population studies revealed strong signatures of natural selection for a 1 kb window covering the SNP 144716 in two human populations, the European American (π = 0.0072, Tajima's D= 3.31, 14 SNPs) and the Japanese (π = 0.0049, Fay & Wu's H = 8.05, 14 SNPs), as well as in chimpanzees (Fay & Wu's H = 8.62, 12 SNPs). These results strongly support the involvement of the FHIT intronic region in an increased risk of prostate cancer. © 2008 Ding et al

Congenital Hydrocephalus and Abnormal Subcommissural Organ Development in Sox3 Transgenic Mice

Congenital hydrocephalus (CH) is a life-threatening medical condition in which excessive accumulation of CSF leads to ventricular expansion and increased intracranial pressure. Stenosis (blockage) of the Sylvian aqueduct (Aq; the narrow passageway that connects the third and fourth ventricles) is a common form of CH in humans, although the genetic basis of this condition is unknown. Mouse models of CH indicate that Aq stenosis is associated with abnormal development of the subcommmissural organ (SCO) a small secretory organ located at the dorsal midline of the caudal diencephalon. Glycoproteins secreted by the SCO generate Reissner's fibre (RF), a thread-like structure that descends into the Aq and is thought to maintain its patency. However, despite the importance of SCO function in CSF homeostasis, the genetic program that controls SCO development is poorly understood. Here, we show that the X-linked transcription factor SOX3 is expressed in the murine SCO throughout its development and in the mature organ. Importantly, overexpression of Sox3 in the dorsal diencephalic midline of transgenic mice induces CH via a dose-dependent mechanism. Histological, gene expression and cellular proliferation studies indicate that Sox3 overexpression disrupts the development of the SCO primordium through inhibition of diencephalic roof plate identity without inducing programmed cell death. This study provides further evidence that SCO function is essential for the prevention of hydrocephalus and indicates that overexpression of Sox3 in the dorsal midline alters progenitor cell differentiation in a dose-dependent manner

An FDA bioinformatics tool for microbial genomics research on molecular characterization of bacterial foodborne pathogens using microarrays

<p>Abstract</p> <p>Background</p> <p>Advances in microbial genomics and bioinformatics are offering greater insights into the emergence and spread of foodborne pathogens in outbreak scenarios. The Food and Drug Administration (FDA) has developed a genomics tool, ArrayTrack^TM, which provides extensive functionalities to manage, analyze, and interpret genomic data for mammalian species. ArrayTrack^TM has been widely adopted by the research community and used for pharmacogenomics data review in the FDA’s Voluntary Genomics Data Submission program. </p> <p>Results</p> <p>ArrayTrack^TM has been extended to manage and analyze genomics data from bacterial pathogens of human, animal, and food origin. It was populated with bioinformatics data from public databases such as NCBI, Swiss-Prot, KEGG Pathway, and Gene Ontology to facilitate pathogen detection and characterization. ArrayTrack^TM’s data processing and visualization tools were enhanced with analysis capabilities designed specifically for microbial genomics including flag-based hierarchical clustering analysis (HCA), flag concordance heat maps, and mixed scatter plots. These specific functionalities were evaluated on data generated from a custom Affymetrix array (FDA-ECSG) previously developed within the FDA. The FDA-ECSG array represents 32 complete genomes of <it>Escherichia coli</it> and<it> Shigella.</it> The new functions were also used to analyze microarray data focusing on antimicrobial resistance genes from <it>Salmonella</it> isolates in a poultry production environment using a universal antimicrobial resistance microarray developed by the United States Department of Agriculture (USDA).</p> <p>Conclusion</p> <p>The application of ArrayTrack^TM to different microarray platforms demonstrates its utility in microbial genomics research, and thus will improve the capabilities of the FDA to rapidly identify foodborne bacteria and their genetic traits (e.g., antimicrobial resistance, virulence, etc.) during outbreak investigations. ArrayTrack^TM is free to use and available to public, private, and academic researchers at <url>http://www.fda.gov/ArrayTrack</url>. </p

Longitudinal Evaluation of an N-Ethyl-N-Nitrosourea-Created Murine Model with Normal Pressure Hydrocephalus

Normal-pressure hydrocephalus (NPH) is a neurodegenerative disorder that usually occurs late in adult life. Clinically, the cardinal features include gait disturbances, urinary incontinence, and cognitive decline.Herein we report the characterization of a novel mouse model of NPH (designated p23-ST1), created by N-ethyl-N-nitrosourea (ENU)-induced mutagenesis. The ventricular size in the brain was measured by 3-dimensional micro-magnetic resonance imaging (3D-MRI) and was found to be enlarged. Intracranial pressure was measured and was found to fall within a normal range. A histological assessment and tracer flow study revealed that the cerebral spinal fluid (CSF) pathway of p23-ST1 mice was normal without obstruction. Motor functions were assessed using a rotarod apparatus and a CatWalk gait automatic analyzer. Mutant mice showed poor rotarod performance and gait disturbances. Cognitive function was evaluated using auditory fear-conditioned responses with the mutant displaying both short- and long-term memory deficits. With an increase in urination frequency and volume, the mutant showed features of incontinence. Nissl substance staining and cell-type-specific markers were used to examine the brain pathology. These studies revealed concurrent glial activation and neuronal loss in the periventricular regions of mutant animals. In particular, chronically activated microglia were found in septal areas at a relatively young age, implying that microglial activation might contribute to the pathogenesis of NPH. These defects were transmitted in an autosomal dominant mode with reduced penetrance. Using a whole-genome scan employing 287 single-nucleotide polymorphic (SNP) markers and further refinement using six additional SNP markers and four microsatellite markers, the causative mutation was mapped to a 5.3-cM region on chromosome 4.Our results collectively demonstrate that the p23-ST1 mouse is a novel mouse model of human NPH. Clinical observations suggest that dysfunctions and alterations in the brains of patients with NPH might occur much earlier than the appearance of clinical signs. p23-ST1 mice provide a unique opportunity to characterize molecular changes and the pathogenic mechanism of NPH

The p53 Inhibitor MDM2 Facilitates Sonic Hedgehog-Mediated Tumorigenesis and Influences Cerebellar Foliation

Disruption of cerebellar granular neuronal precursor (GNP) maturation can result in defects in motor coordination and learning, or in medulloblastoma, the most common childhood brain tumor. The Sonic Hedgehog (Shh) pathway is important for GNP proliferation; however, the factors regulating the extent and timing of GNP proliferation, as well as GNP differentiation and migration are poorly understood. The p53 tumor suppressor has been shown to negatively regulate the activity of the Shh effector, Gli1, in neural stem cells; however, the contribution of p53 to the regulation of Shh signaling in GNPs during cerebellar development has not been determined. Here, we exploited a hypomorphic allele of Mdm2 (Mdm2puro), which encodes a critical negative regulator of p53, to alter the level of wild-type MDM2 and p53 in vivo. We report that mice with reduced levels of MDM2 and increased levels of p53 have small cerebella with shortened folia, reminiscent of deficient Shh signaling. Indeed, Shh signaling in Mdm2-deficient GNPs is attenuated, concomitant with decreased expression of the Shh transducers, Gli1 and Gli2. We also find that Shh stimulation of GNPs promotes MDM2 accumulation and enhances phosphorylation at serine 166, a modification known to increase MDM2-p53 binding. Significantly, loss of MDM2 in Ptch1+/− mice, a model for Shh-mediated human medulloblastoma, impedes cerebellar tumorigenesis. Together, these results place MDM2 at a major nexus between the p53 and Shh signaling pathways in GNPs, with key roles in cerebellar development, GNP survival, cerebellar foliation, and MB tumorigenesis

Looking at Cerebellar Malformations through Text-Mined Interactomes of Mice and Humans

WE HAVE GENERATED AND MADE PUBLICLY AVAILABLE TWO VERY LARGE NETWORKS OF MOLECULAR INTERACTIONS: 49,493 mouse-specific and 52,518 human-specific interactions. These networks were generated through automated analysis of 368,331 full-text research articles and 8,039,972 article abstracts from the PubMed database, using the GeneWays system. Our networks cover a wide spectrum of molecular interactions, such as bind, phosphorylate, glycosylate, and activate; 207 of these interaction types occur more than 1,000 times in our unfiltered, multi-species data set. Because mouse and human genes are linked through an orthological relationship, human and mouse networks are amenable to straightforward, joint computational analysis. Using our newly generated networks and known associations between mouse genes and cerebellar malformation phenotypes, we predicted a number of new associations between genes and five cerebellar phenotypes (small cerebellum, absent cerebellum, cerebellar degeneration, abnormal foliation, and abnormal vermis). Using a battery of statistical tests, we showed that genes that are associated with cerebellar phenotypes tend to form compact network clusters. Further, we observed that cerebellar malformation phenotypes tend to be associated with highly connected genes. This tendency was stronger for developmental phenotypes and weaker for cerebellar degeneration

Маркеры ранних стадий рака легкого у ликвидаторов Чернобыльской аварии

Persons exposed to inhaled radionuclides are at high risk for lung cancer, primarily, due to lung fibrosis development. This work studied lung fibrotic changes and lung cancer genetic markers in liquidators of the Chernobyl accident consequences. The study involved 33 men worked at the Chernobyl station area in 1986-1987, aged from 37 to 65 years (average 45.45±0.95; M±m), and 10 control group men comparable on the age, hazards, social and occupational status, lung pathology, but had never exposed to radiation.Lung function tests, fibreoptic bronchoscopy, histological examination of lung and bronchi tissues biopsy specimens, and lung computed tomography were performed. Oncogene c-myc, Ki-67 proliferation marker, and a deletion in the short arm of the chromosome 3 were investigated in the bronchial epithelium.The lung fibrosis was detected in 13 (42.4%) liquidators and in no-one of the control group. The liquidators also had more bronchial epithelium dysregenerative changes with significant prevalence of basement cell hyperplasia (0.58±.09 and 0.2±0.13 cases in the groups accordingly, p&lt;0.05). Meanwhile the control group had no mild dysplasia at all and demonstrated severe dysplasia rather more frequently (0.2±0.13 vs 0.06±0.04 in the liquidators, p&lt;0.05). The liquidators showed the 3p deletion in the bronchial epithelium cells more often (0.21±0.07 and 0.1±0.1 accordingly, p&lt;0.05) and tended to show other genetic lung cancer markers quite more frequent either.The correlation was found between the lung fibrosis and 3p deletion presence (r=0.45, p&lt;0.05) and also between a pneumonia number anamnestically and Ki-67 expression in the liquidators’ group (r=0.46, p&lt;0.05). The second patients’ group did not demonstrated such the correlaions.Thus, chronic respiratory pathology liquidators develop lung fibrosis earlier than other patients with the similar lung pathology. Basement cell hyperplasia and some oncogenes (3p deletion, c-myc) encounter considerably more often in liquidators that likely to be poor prognostic signs for lung cancer occurrence. Therefore, the Chernobyl accident liquidators are at higher risk for lung cancer compared to the similar lung pathology patients without radiation exposureЛица, ингалировавшие радионуклиды в результате профессиональных воздействий либо неблагоприятной экологической обстановки, имеют высокий риск заболевания раком легкого, в первую очередь, за счет развития пневмофиброза. В данной работе исследовались фиброзные изменения легочной ткани и ряд генетических маркеров рака легкого у ликвидаторов последствий Чернобыльской аварии. В исследование вошли 33 ликвидатора (все мужчины) в возрасте от 37 до 65 лет (в среднем 45,4±0,95 года; М±т) и 10 мужчин группы сравнения, сравнимые с “ликвидаторами” по возрасту, вредным привычкам, социально-бытовым и профессиональным условиям, характеру и длительности бронхолегочной патологии, но не контактировавшие с радиацией.Пациентам проводились исследования легочной функции, фибробронхоскопия с биопсией и последующим гистологическим исследованием ткани бронхов и легких, компьютерная томография легких. В биоптатах определяли онкоген c-myc и маркер пролиферации Ki-67, делецию в коротком плече 3-й хромосомы.Среди ликвидаторов пневмофиброз был выявлен у 13 (42,4%) человек, в группе сравнения не выявлен ни у кого. Также у ликвидаторов чаще, чем в группе сравнения, встречались дисрегенераторные изменения бронхиального эпителия с достоверным преобладанием базальноклеточной гиперплазии (0,58±0,09 и 0,2±0,13 случаев соответственно по группам, р&lt;0,05). Вместе с тем в группе сравнения совсем не обнаруживалась дисплазия 1 ст. и несколько чаще встречалась дисплазия 3 ст. (0,2±0,13 в группе сравнения и 0,06±0,04 у ликвидаторов, р&lt;0,05). Делеция Зр в клетках бронхиального эпителия у ликвидаторов определялась достоверно чаще, чем в группе сравнения (0,21±0,07 и 0,1±0,1 соответственно, р&lt;0,05). Имеется тенденция к более частому обнаружению в группе ликвидаторов и других генетических маркеров.Корреляционный анализ показал достоверную связь между наличием пневмофиброза и делеции в коротком плече 3-й хромосомы (r=0,45, р&lt;0,05), а также между числом перенесенных пневмоний и экспрессией Ki-67 в группе ликвидаторов (r=0,46, р&lt;0,05). В группе сравнения такой зависимости не получено.Таким образом, у ликвидаторов с ХОБЛ фиброз легочной ткани развивается значительно раньше, чем у обычных больных ХОБЛ. У ликвидаторов достоверно чаще обнаруживаются базальноклеточная гиперплазия и генетические маркеры рака легкого (делеция Зр, с-тус), что является прогностически неблагоприятным фактором в развитии этого заболевания. В результате ликвидаторы имеют больший риск заболевания раком легкого по сравнению с обычными больными ХОБЛ.