Diagnosing idiopathic learning disability: a cost-effectiveness analysis of microarray technology in the National Health Service of the United Kingdom

Array based comparative genomic hybridisation (aCGH) is a powerful technique for detecting clinically relevant genome imbalance and can offer 40 to > 1000 times the resolution of karyotyping. Indeed, idiopathic learning disability (ILD) studies suggest that a genome-wide aCGH approach makes 10–15% more diagnoses involving genome imbalance than karyotyping. Despite this, aCGH has yet to be implemented as a routine NHS service. One significant obstacle is the perception that the technology is prohibitively expensive for most standard NHS clinical cytogenetics laboratories. To address this, we investigated the cost-effectiveness of aCGH versus standard cytogenetic analysis for diagnosing idiopathic learning disability (ILD) in the NHS. Cost data from four participating genetics centres were collected and analysed. In a single test comparison, the average cost of aCGH was ￡442 and the average cost of karyotyping was ￡117 with array costs contributing most to the cost difference. This difference was not a key barrier when the context of follow up diagnostic tests was considered. Indeed, in a hypothetical cohort of 100 ILD children, aCGH was found to cost less per diagnosis (￡3,118) than a karyotyping and multi-telomere FISH approach (￡4,957). We conclude that testing for genomic imbalances in ILD using microarray technology is likely to be cost-effective because long-term savings can be made regardless of a positive (diagnosis) or negative result. Earlier diagnoses save costs of additional diagnostic tests. Negative results are cost-effective in minimising follow-up test choice. The use of aCGH in routine clinical practice warrants serious consideration by healthcare providers. Copyright statement The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd, and its Licensees to permit this article (if accepted) to be published in BMJ editions and any other BMJPGL products and to exploit all subsidiary rights, as set out in our licence (bmj.com/advice/copyright.shtml). Authorship The authors included on this paper fulfil the criteria of authorship and no one who fulfils the criteria has been excluded from authorship. The authors made a substantial contribution to the conception, design, analysis and interpretation of data. They were involved in drafting the article or revising it critically for important intellectual content and approving the version to be published. Contributorship Sarah Wordsworth (Guarantor): Planning, conducting and reporting work, interpretation of data, drafting and revising article. James Buchanan: Conducting and reporting work, interpretation of data, revising article. Regina Regan: Completing costing questionnaire, providing protocol details, other costing information, interpretation of data, information about learning disability and genome imbalance and revising article. Val Davison: Completing costing questionnaire, providing protocol details, sharing overall laboratory experience and drafting article. Kim Smith: Completing costing questionnaire, providing protocol details, drafting article. Sara Dyer: Completing costing questionnaire and providing protocol details. Carolyn Campbell: Completing costing questionnaire and providing protocol details. Edward Blair: Critical appraisal of article for clinical content and revising article. Eddy Maher: Completing costing questionnaire, providing protocol details, sharing overall laboratory experience and drafting article. Jenny Taylor: Planning and facilitating work between centres. Drafting and revising article. Samantha JL Knight: Completing costing questionnaire, providing protocol details, other costing information, interpretation of data, providing information about learning disability and genome imbalance, drafting and revising article. Jenny Taylor and Samantha JL Knight contributed equally to the work presented.     

Coxsackievirus entry across epithelial tight junctions requires occludin and the small GTPases Rab34 and Rab5

The major group B coxsackievirus (CVB) receptor is a component of the epithelial tight junction (TJ), a protein complex that regulates the selective passage of ions and molecules across the epithelium. CVB enters polarized epithelial cells from the TJ, causing a transient disruption of TJ integrity. Here we show that CVB does not induce major reorganization of the TJ, but stimulates the specific internalization of occludin-a TJ integral membrane component-within macropinosomes. Although occludin does not interact directly with virus, depletion of occludin prevents CVB entry into the cytoplasm and inhibits infection. Both occludin internalization and CVB entry require caveolin but not dynamin; both are blocked by inhibitors of macropinocytosis and require the activity of Rab34, Ras, and Rab5, GTPases known to regulate macropinocytosis. Thus, CVB entry depends on occludin and occurs by a process that combines aspects of caveolar endocytosis with features characteristic of macropinocytosis.     

Distribution, diversity and ecology of aerobic CO-oxidizing bacteria

Numerous studies indicate that carbon monoxide (CO) participates in a broader range of processes than any other single molecule, ranging from subcellular to planetary scales. Despite its toxicity to many organisms, a diverse group of bacteria that span multiple phylogenetic lineages metabolize CO. These bacteria are globally distributed and include pathogens, plant symbionts and biogeochemically important lineages in soils and the oceans. New molecular and isolation techniques, as well as genome sequencing, have greatly expanded our knowledge of the diversity of CO oxidizers. Here, we present a newly emerging picture of the distribution, diversity and ecology of aerobic CO-oxidizing bacteria.     

Assessing the significance of conserved genomic aberrations using high resolution genomic microarrays

Genomic aberrations recurrent in a particular cancer type can be important prognostic markers for tumor progression. Typically in early tumorigenesis, cells incur a breakdown of the DNA replication machinery that results in an accumulation of genomic aberrations in the form of duplications, deletions, translocations, and other genomic alterations. Microarray methods allow for finer mapping of these aberrations than has previously been possible; however, data processing and analysis methods have not taken full advantage of this higher resolution. Attention has primarily been given to analysis on the single sample level, where multiple adjacent probes are necessarily used as replicates for the local region containing their target sequences. However, regions of concordant aberration can be short enough to be detected by only one, or very few, array elements. We describe a method called Multiple Sample Analysis for assessing the significance of concordant genomic aberrations across multiple experiments that does not require a-priori definition of aberration calls for each sample. If there are multiple samples, representing a class, then by exploiting the replication across samples our method can detect concordant aberrations at much higher resolution than can be derived from current single sample approaches. Additionally, this method provides a meaningful approach to addressing population-based questions such as determining important regions for a cancer subtype of interest or determining regions of copy number variation in a population. Multiple Sample Analysis also provides single sample aberration calls in the locations of significant concordance, producing high resolution calls per sample, in concordant regions. The approach is demonstrated on a dataset representing a challenging but important resource: breast tumors that have been formalin-fixed, paraffin-embedded, archived, and subsequently UV-laser capture microdissected and hybridized to two-channel BAC arrays using an amplification protocol. We demonstrate the accurate detection on simulated data, and on real datasets involving known regions of aberration within subtypes of breast cancer at a resolution consistent with that of the array. Similarly, we apply our method to previously published datasets, including a 250K SNP array, and verify known results as well as detect novel regions of concordant aberration. The algorithm has been fully implemented and tested and is freely available as a Java application at http://www.cbil.upenn.edu/MSA.     

Localization of RHO-4 indicates differential regulation of conidial versus vegetative septation in the filamentous fungus Neurospora crassa

Rho-4 mutants of the filamentous fungus Neurospora crassa lack septa and asexual spores (conidia) and grow slowly. In this report, localization of green fluorescent protein-tagged RHO-4 is used to elucidate the differences in factors controlling RHO-4 localization during vegetative growth versus asexual development. RHO-4 forms a ring at incipient vegetative septation sites that constricts with the formation of the septum toward the septal pore; RHO-4 persists around the septal pore after septum completion. During the formation of conidia, RHO-4 localizes to the primary septum but subsequently is relocalized to the cytoplasm after the placement of the secondary septum. Cytoplasmic localization and inactivation of RHO-4 are mediated by a direct physical interaction with RDI-1, a RHO guanosine nucleotide dissociation inhibitor. Inappropriate activation of the cyclic AMP-dependent protein kinase A pathway during vegetative growth causes mislocalization of RHO-4 away from septa to the cytoplasm, a process which was dependent upon RDI-1. An adenylate cyclase cr-1 mutant partially suppresses the aconidial defect of rho-4 mutants but only rarely suppresses the vegetative septation defect, indicating that conidial septation is negatively regulated by CR-1. These data highlight the differences in the regulation of septation during conidiation versus vegetative septation in filamentous fungi.     

Accurate phylogenetic classification of variable-length DNA fragments

Metagenome studies have retrieved vast amounts of sequence data from a variety of environments leading to new discoveries and insights into the uncultured microbial world. Except for very simple communities, the encountered diversity has made fragment assembly and the subsequent analysis a challenging problem. A taxonomic characterization of metagenomic fragments is required for a deeper understanding of shotgun-sequenced microbial communities, but success has mostly been limited to sequences containing phylogenetic marker genes. Here we present PhyloPythia, a composition-based classifier that combines higher-level generic clades from a set of 340 completed genomes with sample-derived population models. Extensive analyses on synthetic and real metagenome data sets showed that PhyloPythia allows the accurate classification of most sequence fragments across all considered taxonomic ranks, even for unknown organisms. The method requires no more than 100 kb of training sequence for the creation of accurate models of sample-specific populations and can assign fragments >or=1 kb with high specificity.     

Two optimized combination assays to examine apoptosis pathways in clinical samples.

BACKGROUND: A consequence of a number of diseases is an alteration in apoptosis. Currently, there is no single assay that measures the main stages of apoptosis, requiring that multiple assays be performed. This hinders studies on clinical samples that have limited cell numbers. Our objective was to combine and optimize assays that target specific stages of apoptosis for use in a typical clinical blood sample. METHODS: Two flow cytometric assays were developed for use on peripheral blood mononuclear cells (PBMC) collected in two 8-ml tubes from a single draw. One measures caspase-12 activity, the level of active caspase-3 and DNA fragmentation. The second assesses depolarization of the mitochondria and phosphatidylserine externalization. Cell populations present within the samples were determined by flow cytometry. Apoptosis was validated by ELISA. RESULTS: Each assay was optimized for use with cell numbers and sample volumes typical of clinical blood samples. Each combination assay effectively distinguished apoptotic from nonapoptotic blood cells. CONCLUSIONS: This combined optimized method comprised of two independent assays makes it possible to assay the major pathways of apoptosis in addition to determining the blood cell subsets that are affected.     

The molecular mechanism of induction of unfolded protein response by Chlamydia

The unfolded protein response (UPR) contributes to chlamydial pathogenesis, as a source of lipids and ATP during replication, and for establishing the initial anti-apoptotic state of host cell that ensures successful inclusion development. The molecular mechanism(s) of UPR induction by Chlamydia is unknown. Chlamydia use type III secretion system (T3SS) effector proteins (e.g, the Translocated Actin-Recruiting Phosphoprotein (Tarp) to stimulate host cell's cytoskeletal reorganization that facilitates invasion and inclusion development. We investigated the hypothesis that T3SS effector-mediated assembly of myosin-II complex produces activated non-muscle myosin heavy chain II (NMMHC-II), which then binds the UPR master regulator (BiP) and/or transducers to induce UPR. Our results revealed the interaction of the chlamydial effector proteins (CT228 and Tarp) with components of the myosin II complex and UPR regulator and transducer during infection. These interactions caused the activation and binding of NMMHC-II to BiP and IRE1α leading to UPR induction. In addition, specific inhibitors of myosin light chain kinase, Tarp oligomerization and myosin ATPase significantly reduced UPR activation and Chlamydia replication. Thus, Chlamydia induce UPR through T3SS effector-mediated activation of NMMHC-II components of the myosin complex to facilitate infectivity. The finding provides greater insights into chlamydial pathogenesis with the potential to identify therapeutic targets and formulations.     

A novel insertion mutation in atlastin 1 is associated with spastic quadriplegia,increased membrane tethering,and aberrant conformational switching

Hereditary spastic paraplegia (HSP) comprises a heterogeneous group of neuropathies affecting upper motor neurons and causing progressive gait disorder. Mutations in the gene SPG3A/atlastin-1 (ATL1), encoding a dynamin superfamily member, which utilizes the energy from GTP hydrolysis for membrane tethering and fusion to promote the formation of a highly branched, smooth endoplasmic reticulum (ER), account for approximately 10% of all HSP cases. The continued discovery and characterization of novel disease mutations are crucial for our understanding of HSP pathogenesis and potential treatments. Here, we report a novel disease-causing, in-frame insertion in the ATL1 gene, leading to inclusion of an additional asparagine residue at position 417 (N417ins). This mutation correlates with complex, early-onset spastic quadriplegia affecting all four extremities, generalized dystonia, and a thinning of the corpus callosum. We show using limited proteolysis and FRET-based studies that this novel insertion affects a region in the protein central to intramolecular interactions and GTPase-driven conformational change, and that this insertion mutation is associated with an aberrant prehydrolysis state. While GTPase activity remains unaffected by the insertion, membrane tethering is increased, indicative of a gain-of-function disease mechanism uncommon for ATL1-associated pathologies. In conclusion, our results identify a novel insertion mutation with altered membrane tethering activity that is associated with spastic quadriplegia, potentially uncovering a broad spectrum of molecular mechanisms that may affect neuronal function.     

An antitumor promoter from Moringa oleifera Lam.

In the course of studies on the isolation of bioactive compounds from Philippine plants, the seeds of Moringa oleifera Lam. were examined and from the ethanol extract were isolated the new O-ethyl-4-(α- -rhamnosyloxy)benzyl carbamate (1) together with seven known compounds, 4(α- -rhamnosyloxy)-benzyl isothiocyanate (2), niazimicin (3), niazirin (4), β-sitosterol (5), glycerol-1-(9-octadecanoate) (6), 3-O-(6′-O-oleoyl-β- -glucopyranosyl)-β-sitosterol (7), and β-sitosterol-3-O-β- -glucopyranoside (8). Four of the isolates (2, 3, 7, and 8), which were obtained in relatively good yields, were tested for their potential antitumor promoting activity using an in vitro assay which tested their inhibitory effects on Epstein–Barr virus-early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). All the tested compounds showed inhibitory activity against EBV-EA activation, with compounds 2, 3 and 8 having shown very significant activities. Based on the in vitro results, niazimicin (3) was further subjected to in vivo test and found to have potent antitumor promoting activity in the two-stage carcinogenesis in mouse skin using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as tumor promoter. From these results, niazimicin (3) is proposed to be a potent chemo-preventive agent in chemical carcinogenesis.