Set Of Genetic Changes Research Articles

Identification of genetic instability in peripheral blood lymphocyte of oral squamous cell carcinoma patients assess by comet assay.

Context:Studies established that human cancer is principally a genetic disease; it arises as accumulation of a set of genetic changes. In the pathogenesis of cancer, genetic instability is the sequential event to a carcinogenic stimulus resulting in various genomic changes including DNA damage.Aims:To assess genetic instability, as susceptibility to DNA damage, we used single-cell gel electrophoresis (comet assay) to study double strand breaks in associated with the risk of oral squamous cell carcinoma (OSCC).Materials and Methods:We used comet assay to measure double strand break in individual peripheral blood lymphocytes from 50 individuals with OSCC and 30 healthy control subjects. All personal information was gathered from subjects including tobacco history. DNA damage was visualized as comet assay and quantified by movement of damaged strands as length of tail.Results:Study results of OSCC patients were observed in relation to clinical staging and histological grading of carcinoma. On the basis of clinical observation, cases were grouped in to Stage I, Stage II, Stage III and Stage IV. No stage I cases were in study sample. The mean DNA damage migration length was observed 4.600 ± 0.4613 μm in stage II, whereas in Stage III and Stage IV, it was observed to be 4.961 ± 0.5620 μm and 4.883 ± 0.410 μm, respectively. The DNA damage length in histological grades of squamous cell carcinoma patients in Grade I was 4.6437 ± 0.3061 μm and Grade II was 5.3533 ± 0.3831 μm. In comparison with control group and squamous cell carcinoma group, it was observed in the range of 0.02–0.36 μm and varied from 4.04 to 5.84 μm range, respectively. Thus, the results were statistically significant with the histological grading of OSCC.Statistical Analysis:Unpaired' test and “ANOVA” test are used for statistics.Statistical Analysis:Unpaired' test and “ANOVA” test are used for statistics.Conclusion:The amount of DNA strand breaks in peripheral lymphocytes are measured by comet assay which is associated with relative risk of OSCC.

Hearing loss genes reveal patterns of adaptive evolution at the coding and non-coding levels in mammals

BackgroundMammals possess unique hearing capacities that differ significantly from those of the rest of the amniotes. In order to gain insights into the evolution of the mammalian inner ear, we aim to identify the set of genetic changes and the evolutionary forces that underlie this process. We hypothesize that genes that impair hearing when mutated in humans or in mice (hearing loss (HL) genes) must play important roles in the development and physiology of the inner ear and may have been targets of selective forces across the evolution of mammals. Additionally, we investigated if these HL genes underwent a human-specific evolutionary process that could underlie the evolution of phenotypic traits that characterize human hearing.ResultsWe compiled a dataset of HL genes including non-syndromic deafness genes identified by genetic screenings in humans and mice. We found that many genes including those required for the normal function of the inner ear such as LOXHD1, TMC1, OTOF, CDH23, and PCDH15 show strong signatures of positive selection. We also found numerous noncoding accelerated regions in HL genes, and among them, we identified active transcriptional enhancers through functional enhancer assays in transgenic zebrafish.ConclusionsOur results indicate that the key inner ear genes and regulatory regions underwent adaptive evolution in the basal branch of mammals and along the human-specific branch, suggesting that they could have played an important role in the functional remodeling of the cochlea. Altogether, our data suggest that morphological and functional evolution could be attained through molecular changes affecting both coding and noncoding regulatory regions.

Substrate cross-feeding affects the speed and trajectory of molecular evolution within a synthetic microbial assemblage

BackgroundSubstrate cross-feeding occurs when one organism partially consumes a primary substrate into one or more metabolites while other organisms then consume the metabolites. While pervasive within microbial communities, our knowledge about the effects of substrate cross-feeding on microbial evolution remains limited. To address this knowledge gap, we experimentally evolved isogenic nitrite (NO2−) cross-feeding microbial strains together for 700 generations, identified genetic changes that were acquired over the evolution experiment, and compared the results with an isogenic completely denitrifying strain that was evolved alone for 700 generations. We further investigated how the magnitude of interdependence between the nitrite cross-feeding strains affects the main outcomes. Our main objective was to quantify how substrate cross-feeding and the magnitude of interdependence affect the speed and trajectory of molecular evolution.ResultsWe found that each nitrite (NO2−) cross-feeding strain acquired fewer genetic changes than did the completely denitrifying strain. In contrast, pairs of nitrite cross-feeding strains together acquired more genetic changes than did the completely denitrifying strain. Moreover, nitrite cross-feeding promoted population diversification, as pairs of nitrite cross-feeding strains acquired a more varied set of genetic changes than did the completely denitrifying strain. These outcomes likely occurred because nitrite cross-feeding enabled the co-existence of two distinct microbial strains, thus increasing the amount of genetic variation for selection to act upon. Finally, the nitrite cross-feeding strains acquired different types of genetic changes than did the completely denitrifying strain, indicating that nitrite cross-feeding modulates the trajectory of molecular evolution.ConclusionsOur results demonstrate that substrate cross-feeding can affect both the speed and trajectory of molecular evolution within microbial populations. Substrate cross-feeding can therefore have potentially important effects on the life histories of microorganisms.

Optimization Approaches for the In Silico Discovery of Optimal Targets for Gene Over/Underexpression

Metabolic engineering (ME) efforts have been recently boosted by the increase in the number of annotated genomes and by the development of several genome-scale metabolic models for microbes of interest in industrial biotechnology. Based on these efforts, strain optimization methods have been proposed to reach the best set of genetic changes to apply to selected host microbes, in order to create strains that are able to overproduce metabolites of industrial interest. Previous work in strain optimization has been mostly based in finding sets of gene (or reaction) deletions that lead to desired phenotypes in computational simulations. In this work, we focus on enlarging the set of possible genetic changes, considering gene over and underexpression. A gene is considered under (over) expressed if its expression value is constrained to be significantly lower (higher) than the one in the wild-type strain, used as a reference. A method is proposed to propagate relative gene expression values to flux constraints over related reactions, making use of the available transcriptional/translational information. The algorithms chosen for the optimization tasks are metaheuristics such as Evolutionary Algorithms (EA) and Simulated Annealing (SA), based on previous successful work on gene knockout optimization. These methods were modified appropriately to accommodate the novel optimization tasks and were applied to study the optimization of succinic and lactic acid production using Escherichia coli as the host. The results are compared with previous ones obtained in gene knockout optimization, thus showing the usefulness of the approach. The methods proposed in this work were implemented in a novel plug-in for OptFlux, an open-source software framework for ME. Supplementary Material is available at www.liebertonline.com/cmb.

TNFRSF14 and EZH2 Mutations, Chr2p Gain and Copy Number Changes Targeting Genes Whose Proteins Interact with the Microenvironment In Transformed Follicular Lymphoma

AbstractAbstract 799 IntroductionThe accurate prediction and optimal management of transformed follicular lymphoma (t-FL) remains a significant clinical challenge. Investigation of the prognostic significance of genetic factors and the immune microenvironment in FL provides insight into the molecular pathogenesis of transformation and should aid in the development of robust molecular predictors of disease. Patient samplesTo address this issue, molecular profiling was performed on 91 samples including paired FL/t-FL samples from each of 31 patients (with additional germline (n=19) and relapse (n=10) samples where available). Median age at FL diagnosis was 46 years (range 22–71) with a median time to transformation of 4 years (1-16). Median follow-up of surviving patients was 10 years (2-19). Methods and ResultsThe two most frequently mutated genes detected in FL/t-FL were TNFRSF14 (chr1p36.32) and EZH2 (chr7q36.1). Twelve TNFRSF14 mutations were detected in 10 patients (32%) with 3 arising in t-FL and one rendered homozygous on transformation. All were confined to exons 1–6 with 7/12 mutations causing premature termination of the protein. Heterozygous non-synonymous mutations in codon 641 of EZH2 occurred in 9 patients (29%) with 2 arising upon transformation. In mutated cases, the TNFRSF14 and EZH2 loci were subject to recurrent copy number (CN) loss/uniparental disomy (6/12) and gain (6/9) respectively. Genome-wide profiling was performed using the Affymetrix SNP 6.0 array to search for co-operating genetic events. A total of 1120 CN aberrations (CNAs) were identified across the cohort with a median of 5 gains (range 0–13) and 7 losses (0-42) in the earliest available FL (FL1) samples and 8 gains (0-33) and 10 losses (4-32) in the initial t-FL (t-FL1) samples. Recurrent CNAs included loss of chr1p, 6q, 9p, 17p and gain of 1q, 6p, 18 and × in both FL1 and t-FL1.In 18 cases, FL1 samples had CN changes absent from subsequent relapsed FL/t-FL samples. This is consistent with t-FL evolving from a common progenitor cell (CPC). Divergent changes seen in both FL and t-FL at relapse after t-FL1 further augment this hypothesis. CNAs common among different samples identify early events in FL/t-FL development. Recurrent regions of loss and gain affecting ≥20% of cases ranged in size from 4 kb to 60 Mb. Of these, gain 2p16.1-p15 (including REL and BCL11A) in FL1, predicted worse clinical outcome (survival from FL diagnosis; logrank p = 0.004, hazard ratio = 7). No cases with gain 2p16.1-p15, gain 8q24.13-q24.3, del 9p21.3 or gain 18q21.2-q21.33 were alive. Novel CNAs included gain 16p12.1 (containing IL4-R, IL21-R, NSMCE1) in 5 t-FL1 cases, recurrent deletion of ETS1 (n=4) on chr11q24.3 and further micro-deletions targeting CREBBP (chr16p13.3) and ALK (chr2p23.2). ConclusionsThe high mutational frequency of TNFRSF14 together with recurrent gain of IL4-R suggests a link between intra-cellular events and B-T cell interactions in the microenvironment. Enrichment of EZH2 mutations, the prognostic impact of gain REL/BCL11a and recurrent CN changes in CPC cases complete a core set of genetic changes that are potential molecular markers in t-FL. Disclosures:No relevant conflicts of interest to declare.

Presence of Alternative Lengthening of Telomeres Mechanism in Patients With Glioblastoma Identifies a Less Aggressive Tumor Type With Longer Survival

Patients with glioblastoma (GBM) have variable clinical courses, but the factors that underlie this heterogeneity are not understood. To determine whether the presence of the telomerase-independent alternative lengthening of telomeres (ALTs) mechanism is a significant prognostic factor for survival, we performed a retrospective analysis of 573 GBM patients. The presence of ALT was identified in paraffin sections using a combination of immunofluorescence for promyelocytic leukemia body and telomere fluorescence in situ hybridization. Alternative lengthening of telomere was present in 15% of the GBM patients. Patients with ALT had longer survival that was independent of age, surgery, and other treatments. Mutations in isocitrate dehydrogenase (IDH1mut) 1 frequently accompanied ALT, and in the presence of both molecular events, there was significantly longer overall survival. These data suggest that most ALT+ tumors may be less aggressive proneural GBMs, and the better prognosis may relate to the set of genetic changes associated with this tumor subtype. Despite improved overall survival of patients treated with the addition of chemotherapy to radiotherapy and surgery, ALT and chemotherapy independently provided a survival advantage, but these factors were not found to be additive. These results suggest a critical need for developing new therapies to target these specific GBM subtypes.

Programming cells by multiplex genome engineering and accelerated evolution

The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.

POPULATION STRUCTURE, LEVELS OF SELECTION, AND THE EVOLUTION OF INTRACELLULAR SYMBIONTS

Many obligately intracellular symbionts exhibit a characteristic set of genetic changes that include an increase in substitution rates, loss of many genes, and apparent destabilization of many proteins and structural RNAs. Authors have suggested that these changes are due to increased mutation rates, or, more commonly, decreased effective population size due to population bottlenecks at the symbiont or, perhaps, host level. I propose that the increase in substitution rates and accumulation of deleterious mutations is a consequence of the population structure imposed on the endosymbionts by strict host association, loss of horizontal transmission and potentially conflicting levels of selection. I analyze a population genetic model of endosymbiont evolution, and demonstrate that substitution rates will increase, and the effect of those substitutions on endosymbiont fitness will become more deleterious as horizontal transmission among hosts decreases. Additionally, I find that there is a critical level of horizontal transmission below which natural selection cannot effectively purge deleterious mutations, leading to an expected loss of fitness over time. This critical level varies across loci with the degree of correlation between host and endosymbiont fitness, and may help explain differential retention and loss of certain genes.

Integrative Genomic Approaches Identify IKBKE as a Breast Cancer Oncogene

The karyotypic chaos exhibited by human epithelial cancers complicates efforts to identify mutations critical for malignant transformation. Here we integrate complementary genomic approaches to identify human oncogenes. We show that activation of the ERK and phosphatidylinositol 3-kinase (PI3K) signaling pathways cooperate to transform human cells. Using a library of activated kinases, we identify several kinases that replace PI3K signaling and render cells tumorigenic. Whole genome structural analyses reveal that one of these kinases, IKBKE (IKKepsilon), is amplified and overexpressed in breast cancer cell lines and patient-derived tumors. Suppression of IKKepsilon expression in breast cancer cell lines that harbor IKBKE amplifications induces cell death. IKKepsilon activates the nuclear factor-kappaB (NF-kappaB) pathway in both cell lines and breast cancers. These observations suggest a mechanism for NF-kappaB activation in breast cancer, implicate the NF-kappaB pathway as a downstream mediator of PI3K, and provide a framework for integrated genomic approaches in oncogene discovery.

Differential Requirement for RhoA GTPase Depending on the Cellular Localization of Protein Kinase D

This study explores the links between the GTPase RhoA and the serine kinase protein kinase D (PKD) during thymocyte development. The rationale is that RhoA and PKD regulate common biological responses during T cell development, but there is nothing known about their interdependence. In fibroblasts, Rho function is required for activation of PKD catalytic activity. However, the data show that activation of Rho is neither sufficient nor essential for PKD activation in T cells. One alternative explanation for the apparent convergence of PKD and Rho signaling in T cells is that PKD responses might be Rho-dependent. To address this latter possibility, we probed the Rho requirements for the actions of constitutively active PKD mutants in pre-T cells of transgenic mice. Active PKD can localize to either the plasma membrane or the cytosol, and we therefore compared the Rho requirements for the actions of membrane- or cytosol-localized PKD. Here we show that membrane-localized PKD regulation of pre-T cell differentiation is Rho-dependent, but the actions of cytosol-localized PKD are not. These studies demonstrate that a Rho requirement for PKD activation is not ubiquitous. Moreover, links between PKD and Rho are determined by the cellular location of PKD.

The genetics ofradiation-induced and sporadic osteosarcoma: a unifyingtheory?

Cancer is a disease of the genome, with the neoplastic phenotypebeing passed from one cell generation to the other.Radiation-induced cancer has often been considered to representa unique entity amongst neoplasia, with the energy depositionbeing held responsible for both direct (gene mutations) andindirect (bystander effects, induced instability etc) alterations to the cellular genome. However, radiogenic tumoursin man and experimental animals appear to be physiologicallyand genetically indistinguishable from their sporadiccounterparts, suggesting that the aetiologies of these two tumourtypes are in fact closely related. We have conducted a general screen of the genetic alterations inradiation-induced mouse osteosarcoma, a tumour that ishistopathologically indistinguishable from human sporadicosteosarcoma. Comparison of the two tumour types indicates theexistence of a common set of genetic changes, providingadditional evidence to support the concept that the molecular pathology of radiation-induced malignancy is no different tothat of sporadic cancers.