A Systems Biology Interpretation of Array Comparative Genomic Hybridization (aCGH) Data through Phylogenetics

Array Comparative Genomic Hybridization (aCGH) is a rapid screening technique to detect gene deletions and duplications, providing an overview of chromosomal aberrations throughout the entire genome of a tumor, without the need for cell culturing. However, the heterogeneity of aCGH data obfuscates existing methods of data analysis. Analysis of aCGH data from a systems biology perspective or in the context of total aberrations is largely absent in the published literature. We present here a novel alternative to the functional analysis of aCGH data using the phylogenetic paradigm that is well-suited to high dimensional datasets of heterogeneous nature, but has not been widely adapted to aCGH data. Maximum parsimony phylogenetic analysis sorts out genetic data through the simplest presentation of the data on a cladogram, a graphical evolutionary tree, thus providing a powerful and efficient method for aCGH data analysis. For example, the cladogram models the multiphasic changes in the cancer genome and identifies shared early mutations in the disease progression, providing a simple yet powerful means of aCGH data interpretation. As such, applying maximum parsimony phylogenetic analysis to aCGH results allows for the differentiation between drivers and passenger genes aberrations in cancer specimens. In addition to offering a novel methodology to analyze aCGH results, we present here a crucial software suite that we wrote to carry out the analysis. In a broader context, we wish to underscore that phylogenetic analysis of aCGH data is a non-parametric method that circumvents the pitfalls and frustrations of standard analytical techniques that rely on parametric statistics. Organizing the data in a cladogram as explained in this research article provides insights into the disease common aberrations, as well as the disease subtypes and their shared aberrations (the synapomorphies) of each subtype. Hence, we report the method and make the software suite publicly and freely available at http://software.phylomcs.com so that researchers can test alternative and innovative approaches to the analysis of aCGH data

Association of Inherited Thrombophilia with Recurrent Pregnancy Loss in Palestinian Women

Objective. This study aimed at analyzing the association between recurrent pregnancy loss (RPL) and factor V G1691A (FVL), prothrombin G20210 (FII); and MTHFR C677T (MTHFR) in Palestinian women. Method. We studied 329 Palestinian women with RPL and/or stillbirth (SB); and compared them to 402 healthy reproductive Palestinian women. Cases and controls were tested for the above mutations. Odds ratio (OR) at confidence interval (CI) of 95% was used as a measure of association between the mutations and RPL. Results. Our statistical analysis showed a slightly increased association, which was not significant between FVL and RPL (OR 1.32, 95% CI 0.90–1.94), and no association between FII (OR 0.84, 95% CI 0.38–1.92), MTHFR (OR 0.58, 95% CI 0.32–1.03), and RPL. Further analysis of RPL subgroups revealed an association between FVL and first-trimester loss (OR 1.33, 95% CI 0.892–1.989), and second-trimester loss (OR 1.13, 95% CI 0.480–2.426), both were not statistically significant. Furthermore, the only statistically significant association was between FVL and SB (OR 2.0, 95% CI 1.05–3.70). Conclusion. Our analysis had failed to find a significant association between FVL, FII, MTHFR; and RPL in either the first or second trimester. FVL was significantly associated with fetal loss if the loss was a stillbirth

Homozygous frameshift mutations in FAT1 cause a syndrome characterized by colobomatous-microphthalmia, ptosis, nephropathy and syndactyly

A failure in optic fissure fusion during development can lead to blinding malformations of the eye. Here, we report a syndrome characterized by facial dysmorphism, colobomatous microphthalmia, ptosis and syndactyly with or without nephropathy, associated with homozygous frameshift mutations in FAT1. We show that Fat1 knockout mice and zebrafish embryos homozygous for truncating fat1a mutations exhibit completely penetrant coloboma, recapitulating the most consistent developmental defect observed in affected individuals. In human retinal pigment epithelium (RPE) cells, the primary site for the fusion of optic fissure margins, FAT1 is localized at earliest cell-cell junctions, consistent with a role in facilitating optic fissure fusion during vertebrate eye development. Our findings establish FAT1 as a gene with pleiotropic effects in human, in that frameshift mutations cause a severe multi-system disorder whereas recessive missense mutations had been previously associated with isolated glomerulotubular nephropathy

Visualization and Curve-Parameter Estimation Strategies for Efficient Exploration of Phenotype Microarray Kinetics

The Phenotype MicroArray (OmniLog® PM) system is able to simultaneously capture a large number of phenotypes by recording an organism's respiration over time on distinct substrates. This technique targets the object of natural selection itself, the phenotype, whereas previously addressed '-omics' techniques merely study components that finally contribute to it. The recording of respiration over time, however, adds a longitudinal dimension to the data. To optimally exploit this information, it must be extracted from the shapes of the recorded curves and displayed in analogy to conventional growth curves.The free software environment R was explored for both visualizing and fitting of PM respiration curves. Approaches using either a model fit (and commonly applied growth models) or a smoothing spline were evaluated. Their reliability in inferring curve parameters and confidence intervals was compared to the native OmniLog® PM analysis software. We consider the post-processing of the estimated parameters, the optimal classification of curve shapes and the detection of significant differences between them, as well as practically relevant questions such as detecting the impact of cultivation times and the minimum required number of experimental repeats.We provide a comprehensive framework for data visualization and parameter estimation according to user choices. A flexible graphical representation strategy for displaying the results is proposed, including 95% confidence intervals for the estimated parameters. The spline approach is less prone to irregular curve shapes than fitting any of the considered models or using the native PM software for calculating both point estimates and confidence intervals. These can serve as a starting point for the automated post-processing of PM data, providing much more information than the strict dichotomization into positive and negative reactions. Our results form the basis for a freely available R package for the analysis of PM data

Inhibition of IL-10 Production by Maternal Antibodies against Group B Streptococcus GAPDH Confers Immunity to Offspring by Favoring Neutrophil Recruitment

Group B Streptococcus (GBS) is the leading cause of neonatal pneumonia, septicemia, and meningitis. We have previously shown that in adult mice GBS glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an extracellular virulence factor that induces production of the immunosuppressive cytokine interleukin-10 (IL-10) by the host early upon bacterial infection. Here, we investigate whether immunity to neonatal GBS infection could be achieved through maternal vaccination against bacterial GAPDH. Female BALB/c mice were immunized with rGAPDH and the progeny was infected with a lethal inoculum of GBS strains. Neonatal mice born from mothers immunized with rGAPDH were protected against infection with GBS strains, including the ST-17 highly virulent clone. A similar protective effect was observed in newborns passively immunized with anti-rGAPDH IgG antibodies, or F(ab')2 fragments, indicating that protection achieved with rGAPDH vaccination is independent of opsonophagocytic killing of bacteria. Protection against lethal GBS infection through rGAPDH maternal vaccination was due to neutralization of IL-10 production soon after infection. Consequently, IL-10 deficient (IL-10−/−) mice pups were as resistant to GBS infection as pups born from vaccinated mothers. We observed that protection was correlated with increased neutrophil trafficking to infected organs. Thus, anti-rGAPDH or anti-IL-10R treatment of mice pups before GBS infection resulted in increased neutrophil numbers and lower bacterial load in infected organs, as compared to newborn mice treated with the respective control antibodies. We showed that mothers immunized with rGAPDH produce neutralizing antibodies that are sufficient to decrease IL-10 production and induce neutrophil recruitment into infected tissues in newborn mice. These results uncover a novel mechanism for GBS virulence in a neonatal host that could be neutralized by vaccination or immunotherapy. As GBS GAPDH is a structurally conserved enzyme that is metabolically essential for bacterial growth in media containing glucose as the sole carbon source (i.e., the blood), this protein constitutes a powerful candidate for the development of a human vaccine against this pathogen

Somatic Mutations in UBA1 and Severe Adult-Onset Autoinflammatory Disease

BACKGROUND Adult-onset inflammatory syndromes often manifest with overlapping clinical features. Variants in ubiquitin-related genes, previously implicated in autoinflammatory disease, may define new disorders. METHODS We analyzed peripheral-blood exome sequence data independent of clinical phenotype and inheritance pattern to identify deleterious mutations in ubiquitin-related genes. Sanger sequencing, immunoblotting, immunohistochemical testing, flow cytometry, and transcriptome and cytokine profiling were performed. CRISPR-Cas9–edited zebrafish were used as an in vivo model to assess gene function. RESULTS We identified 25 men with somatic mutations affecting methionine-41 (p.Met41) in UBA1, the major E1 enzyme that initiates ubiquitylation. (The gene UBA1 lies on the X chromosome.) In such patients, an often fatal, treatment-refractory inflammatory syndrome develops in late adulthood, with fevers, cytopenias, characteristic vacuoles in myeloid and erythroid precursor cells, dysplastic bone marrow, neutrophilic cutaneous and pulmonary inflammation, chondritis, and vasculitis. Most of these 25 patients met clinical criteria for an inflammatory syndrome (relapsing polychondritis, Sweet’s syndrome, polyarteritis nodosa, or giant-cell arteritis) or a hematologic condition (myelodysplastic syndrome or multiple myeloma) or both. Mutations were found in more than half the hematopoietic stem cells, including peripheral-blood myeloid cells but not lymphocytes or fibroblasts. Mutations affecting p.Met41 resulted in loss of the canonical cytoplasmic isoform of UBA1 and in expression of a novel, catalytically impaired isoform initiated at p.Met67. Mutant peripheral-blood cells showed decreased ubiquitylation and activated innate immune pathways. Knockout of the cytoplasmic UBA1 isoform homologue in zebrafish caused systemic inflammation. CONCLUSIONS Using a genotype-driven approach, we identified a disorder that connects seemingly unrelated adult-onset inflammatory syndromes. We named this disorder the VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. (Funded by the NIH Intramural Research Programs and the EU Horizon 2020 Research and Innovation Program.

A pleiomorphic GH pituitary adenoma from a Carney complex patient displays universal allelic loss at the protein kinase A regulatory subunit 1A (PRKARIA) locus

Carney complex (CNC) is a familial multiple endocrine neoplasia syndrome associated with GH-producing pituitary tumours and transmitted as an autosomal dominant trait. Mutations of the PRKAR1A gene are responsible for approximately half the known CNC cases but have never found in sporadic pituitary tumours. Pituitary tissue was obtained from an acromegalic CNC patient heterozygote for a common (PRKARIA)(i)-inactivating mutation. Both immunohistochemistry and electron microscopy showed a highly pleiomorphic pituitary adenoma. The cell culture population appeared morphologically heterogeneous and remained so after more than 30 passages. The mixture was comprised of cells strongly immunostained for GH, spindle-shaped myofibroblast-like cells, and cuboid cells with large axonal projections (negative for GH). The population appeared to have both epithelial and mesenchymal cells. Both at baseline and at passage 30, cytogenetic analysis indicated the presence of normal 46, XY diploid karyotype, whereas losses of the PRKARIA(i) locus were demonstrated in more than 98% of the cells by fluorescent in situ hybridisation, supporting this gene's involvement in pituitary tumorigenesis. Allelic loss may have occurred in a single precursor cell type that differentiated and clonally expanded into several phenotypes. Epithelial-to-mesenchymal transition may also occur in CNC-associated pleiomorphic pituitary adenomas

Taxonomic significance of foliar epidermal morphology in Lamiaceae from Pakistan

Foliar micromorphological features are useful to elucidate the taxonomy and systematics of the Lamiaceae species. Leaf epidermal morphology using scanning electron microscopy and light microscopy of 22 Lamiaceae species from 15 genera have been investigated with an aim to solve its taxonomic problem in the correct identification. Various foliar micromorphological features were observed to explain their importance in resolving the correct identification of Lamiaceae taxa. Two main types of trichomes were observed; glandular trichomes (GTs) and nonglandular trichomes (NGTs). GTs were further divided into seven subtypes including the capitate, subsessile capitate, sessile capitate, sunken, barrel, peltate, and clavate. Similarly, NGTs were also divided into simple unicellular and multicellular including conical, falcate, cylindrical, dendrite, papillose, and short hook shape. Quantitative measurement includes the length and width of the trichomes, stomatal complex, epidermal cells, stomata, and trichomes index. Based on the foliar micromorphological characters, a taxonomic key was developed to delimit and correctly identify studied taxa. Further molecular, other anatomical and phylogenetic studies are recommended to strengthen the systematics of Lamiaceae