Screening Of Malaysian Patients With Nasopharyngeal Carcinoma (NPC) Using Comparative Genomic Hybridization (CGH) Technique For Identification Of Specific Genetic Alteration

Nasopharyngeal Carcinoma (NPC) is a malignancy that originates from the epithelial lining of Fossa of Rosen Muller in the nasopharynx. It is a highly prevalent disease in Southern China and Southeast Asia including Malaysia. In this study, the genetic alterations from forty-four NPC patients from five hospitals in Malaysia were screened. Comparative Genomic Hybridization (CGH) technique was used for screening the genetic alterations. CGH is a molecular cytogenetic technique which is used to identify imbalanced genetic alterations that is present in this malignant disease

Pharmacological screening using an FXN-EGFP cellular genomic reporter assay for the therapy of Friedreich ataxia

Copyright @ 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol.Muscular Dystrophy Association (USA), the National Health and Medical Research Council (Australia), the Friedreich’s Ataxia Research Alliance (USA), the Brockhoff Foundation (Australia), the Friedreich Ataxia Research Association (Australasia), Seek A Miracle (USA) and the Victorian Government’s Operational Infrastructure Support Program

Análisis de variación estructural en cáncer mediante secuenciación de lecturas largas

Trabajo Fin de Máster en Bioinformática y Biología ComputacionalEl cáncer es una enfermedad compleja, originada como resultado de cambios que pueden darse a distintos niveles del genoma celular. Los avances en las técnicas destinadas a su estudio han abierto las puertas al conocimiento de las mutaciones y los genes implicados en el proceso cancerígeno. Entre ellas, las tecnologías de secuenciación de segunda generación han desempeñado un papel fundamental, al ser capaces de secuenciar el genoma completo de las células presentes en la muestra de un tumor particular. Esto ha permitido la construcción de estándares de referencia tumorales, tanto somáticos como germinales, aunque las mutaciones somáticas constituyen la causa predominante de su desarrollo. Durante los últimos años, las tecnologías de secuenciación de tercera generación han surgido como solución a algunas de las limitaciones intrínsecas de las anteriores. Entre otras, la capacidad de generar lecturas de kilobases a megabases de longitud ha permitido estudiar de forma adecuada regiones del genoma humano más desconocidas (ricas en GC y repetitivas), cuyas características impedían su correcta resolución con lecturas cortas. Estas regiones suponen una fuente de generación activa de ciertos tipos de variantes, de forma que al menos un 70 % de las variantes estructurales del genoma humano había resultado difícil de caracterizar con lecturas cortas. El desarrollo reciente de estas tecnologías de tercera generación presenta la necesidad de describir sus competencias y obstáculos, de lograr su optimización y de desarrollar aproximaciones bioinformáticas para aprovechar al máximo su potencial. Con el objetivo de estudiar las capacidades y limitaciones concretas de las tecnologías de secuenciación de ambas generaciones en la detección y el estudio de variantes estructurales en cáncer, este proyecto ha trabajado sobre datos de secuenciación de genoma completo de las líneas celulares COLO829 y COLO829BL (un tipo de melanoma y su contraparte sana). Para ello, se ha desarrollando un flujo de trabajo utilizando cada una de las dos generaciones de tecnología. Respecto al flujo de trabajo desarrollado basado en lecturas cortas, éste permitió identificar más de un 90 % de las variantes presentes en la referencia somática disponible para COLO829. El flujo de trabajo basado en lecturas largas permitió por su parte identificar más de 30 000 variantes de tipo indel y estructurales. Los resultados obtenidos en este trabajo no sólo han contribuido a demostrar y reforzar el papel clave de las regiones repetitivas en la generación de los tipos de variantes de interés, sino también a la detección específica de tipos complejos. De las variantes identificadas con lecturas largas, destaca una de significancia clínica contrastada en el supresor tumoral PTEN. También resultan interesantes otras dos que, aunque no disponen del mismo estatus, permiten plantear hipótesis sobre el cáncer de estudio, al afectar a genes participantes en rutas de potencial relevancia (TP53TG3B, RPH3AL). De hecho, entre las variantes identificadas y sometidas a una filtración preliminar, 558 de ellas afectan a 243 genes implicados en el desarrollo del cáncer, de forma que las posibles hipótesis a plantear y corroborar podrían ser muchas más. Finalmente, los cruces entre las variantes identificadas por cada flujo de trabajo permitieron proponer nuevas variantes a validar e incluir en la referencia somática de COLO829. Además, entre ellas se encontraron algunas que afectan a 8 genes implicados en el desarrollo cancerígeno

Genes: Multigene Families, Control of Gene Expression, Genetic contributions to Human Diseases, including Chromosomal Fragile Sites and ‘Dynamic’ and ‘Non-self’ Mutations

The early work in this thesis utilizes the general approach of comparative analysis. In order to find out the relationship between entities (either functional or genetic) my colleagues and I have attempted to identify the important elements by detecting similarity between those entities that act in a similar manner. The philosophy behind this approach is simply that when two distinct objects perform a similar process then the requirements essential for that process will be revealed as similarities between those objects above a noise of difference between them. The use of comparative analysis in biological systems is an attempt to identify natural order from apparent chaos. This work includes but is not limited to :- 1. discovery of the family of kallikrein genes and exploration of their roles in biology, 2. identification of the DNA sequence elements required for hormonal and heavy metal control of metallothionein gene expression 3. discovery of at least some of the necessary and sufficient conditions for the appearance of fragile sites on chromosomes, and their consequent contributions to disease, 4. the molecular properties of repeat DNA sequence expansion that lead to dynamic mutation and consequent fragile site expression and / or disease pathogenesis. In a sense the use of genetic animal models in order to study gene function and pathogenesis follows similar logic of comparative analysis – the mutation of a single endogenous gene or the expression of a single introduced mutated gene in a (presumed) constant genetic background to enable the biological consequences of the genetic mutation or aberrant gene expression by comparing animals from the ‘wild-type’ or parent line with those that now carry the mutation or altered gene. This approach has been utilized in the most recent work contained herein as a means to determine gene function and / or to model human genetic disease pathogenesis, specifically pathogenic mechanisms of the protein WWOX in cancer and expanded repeat RNAs in neurodegenerative diseases. The culmination of this recent work is the development of an hypothesis – 4. that expanded repeat double-stranded RNA leads to neurodegeneration through its recognition by the RNA-binding pattern recognition receptors as a ‘non-self’ or foreign nucleic acid due to a paucity of RNA modification. The resultant pathogenic mechanism is therefore autoinflammatory disease. Given the wide range and variety of evidence of inflammatory activation in neurodegenerative diseases in general, this mechanism is therefore hypothesized to be the general causal mechanism for most (or all) of these diseases. A specific Introduction - highlighting the nature and significance of the work, and a Conclusion – of how this work has contributed to knowledge, are given at the start of each chapter, while the impact of the various components of this work is indicated by the number of citations for each of the included publications. Authorship contributions to each of the included publications in this work are also indicated with each specific reference.Thesis (DSc) -- University of Adelaide, School of Biological Sciences, 202

High throughput screening.

<p>The HeLa (<i>FXN-EGFP</i>) stable cell line was used in high throughput screening procedures to screen the Spectrum Collection compound library. Each test compound was evaluated at a final concentration of 10 µM. Controls included untreated cells and cells exposed to 3.3 µM cisplatin. Cultures were incubated for 72 hours. The fluorometric measurement of EGFP fluorescence was performed followed by the fluorometric measurement of cell viability. The average background fluorescence of parental HeLa cells was subtracted from the EGFP readings of HeLa (<i>FXN-EGFP</i>) cells exposed to the chemical library. A correction factor for the number of viable cells was applied to the EGFP levels in each corresponding well. This was compared to the average value obtained from cells not exposed to any test compound to determine the level of change in <i>FXN</i> gene expression.</p

Characterization of HeLa (<i>FXN-EGFP</i>) stable cell lines.

<p>(A) Diagrammatic representation of the BAC genomic DNA fragment containing the <i>FXN-EGFP</i> genomic reporter construct. The sequence includes exons 13–16 of the <i>PIP5K1B</i> gene and the <i>PRKACG</i> gene upstream of the <i>FXN</i> locus, and about 23 kb of additional sequence downstream of exon 5b. The exon 5a–EGFP–Kan/Neo region is shown in greater detail. (B) Microscopic imaging. Transmitted light (left) and fluorescence (right) images of a HeLa (<i>FXN-EGFP</i>) stable cell line. EGFP expression produced by the <i>FXN-EGFP</i> genomic reporter is evident in all cells. (C) Flow cytometric analysis. The levels of EGFP expression (left Y-axis) and the proportion of EGFP-positive cells (right Y-axis) were stable following growth in continuous culture. (D) Determination of transgenic fragment integration site by FISH. Rhodamine-labeled RP11-265B8 was hybridized onto metaphase chromosomes (DAPI stained) of HeLa (left) and HeLa (<i>FXN-EGFP</i>) (right) cells. Three hybridization signals (yellow arrows) corresponded to the endogenous <i>FXN</i> gene. The presence of one additional brighter signal (orange arrow) establishes the presence of a single integration site containing multiple copies of the <i>FXN-EGFP</i> transgene.</p