Brca1 and brca2:Role in the DNA damage response, cancer formation and treatment

BRCA1 and BRCA2 are highly penetrant breast and ovarian cancer susceptibility genes that are mutated in a significant proportion of familial breast and ovarian cancer syndromes. Both of these genes are tumour suppressors, the products of which play vital roles in the cellular response to DNA damage. These proteins function in a number of cellular pathways in order to maintain genomic stability including DNA damage signaling, DNA repair, cell cycle regulation, protein ubiquitination, chromatin remodeling, transcriptional regulation and apoptosis. This chapter will discuss the functions of these proteins and how they relate to tumour development, and therapy. © 2009 Springer Science+Business Media B.V.

DNA mismatch repair and the transition to hormone independence in breast and prostate cancer

The molecular basis for the progression of breast and prostate cancer from hormone dependent to hormone independent disease remains a critical issue in the management of these two cancers. The DNA mismatch repair system is integral to the maintenance of genomic stability and suppression of tumorigenesis. No firm consensus exists regarding the implications of mismatch repair (MMR) deficiencies in the development of breast or prostate cancer. However, recent studies have reported an association between mismatch repair deficiency and loss of specific hormone receptors, inferring a potential role for mismatch repair deficiency in this transition. An updated review of the experimental data supporting or contradicting the involvement of MMR defects in the development and progression of breast and prostate cancer will be provided with particular emphasis on their implications in the transition to hormone independence.

Variation in DNA repair genes XRCC3, XRCC4, XRCC5 and susceptibility to myeloma

Cytogenetic analysis in myeloma reveals marked chromosomal instability. Both widespread genomic alterations and evidence of aberrant class switch recombination, the physiological process that regulates maturation of the antibody response, implicate the DNA repair pathway in disease pathogenesis. We therefore assessed 27 SNPs in three genes (XRCC3, XRCC4 and XRCC5) central to DNA repair in patients with myeloma and controls from the EpiLymph study and from an Irish hospital registry (n = 306 cases, 263 controls). For the haplotype-tagging SNP (htSNP) rs963248 in XRCC4, Allele A was significantly more frequent in cases than in controls (86.4 versus 80.8%; odds ratio 1.51; 95% confidence interval 1.10-2.08; P = 0.0133), as was the AA genotype (74 versus 65%) (P = 0.026). Haplotype analysis was performed using Unphased for rs963248 in combination with additional SNPs in XRCC4. The strongest evidence of association came from the A-T haplotype from rs963248-rs2891980 (P = 0.008). For XRCC5, the genotype GG from rs1051685 was detected in 10 cases from different national populations but in only one control (P = 0.015). This SNP is located in the 3'-UTR of XRCC5. Overall, these data provide support for the hypothesis that common variation in the genes encoding DNA repair proteins contributes to susceptibility to myeloma.

Maintaining and reprogramming genomic androgen receptor activity in prostate cancer

Prostate cancer treatment is dominated by strategies to control androgen receptor (AR) activity. AR has an impact on prostate cancer development through the regulation of not only transcription networks but also genomic stability and DNA repair, as manifest in the emergence of gene fusions. Whole-genome maps of AR binding sites and transcript profiling have shown changes in the recruitment and regulatory effect of AR on transcription as prostate cancer progresses. Defining other factors that are involved in this reprogramming of AR function gives various opportunities for cancer detection and therapeutic intervention.

Mapping protein-DNA interactions using ChIP-sequencing

Chromatin immunoprecipitation (ChIP) allows enrichment of genomic regions which are associated with specific transcription factors, histone modifications, and indeed any other epitopes which are present on chromatin. The original ChIP methods used site-specific PCR and Southern blotting to confirm which regions of the genome were enriched, on a candidate basis. The combination of ChIP with genomic tiling arrays (ChIP-chip) allowed a more unbiased approach to map ChIP-enriched sites. However, limitations of microarray probe design and probe number have a detrimental impact on the coverage, resolution, sensitivity, and cost of whole-genome tiling microarray sets for higher eukaryotes with large genomes. The combination of ChIP with high-throughput sequencing technology has allowed more comprehensive surveys of genome occupancy, greater resolution, and lower cost for whole genome coverage. Herein, we provide a comparison of high-throughput sequencing platforms and a survey of ChIP-seq analysis tools, discuss experimental design, and describe a detailed ChIP-seq method.Chromatin immunoprecipitation (ChIP) allows enrichment of genomic regions which are associated with specific transcription factors, histone modifications, and indeed any other epitopes which are present on chromatin. The original ChIP methods used site-specific PCR and Southern blotting to confirm which regions of the genome were enriched, on a candidate basis. The combination of ChIP with genomic tiling arrays (ChIP-chip) allowed a more unbiased approach to map ChIP-enriched sites. However, limitations of microarray probe design and probe number have a detrimental impact on the coverage, resolution, sensitivity, and cost of whole-genome tiling microarray sets for higher eukaryotes with large genomes. The combination of ChIP with high-throughput sequencing technology has allowed more comprehensive surveys of genome occupancy, greater resolution, and lower cost for whole genome coverage. Herein, we provide a comparison of high-throughput sequencing platforms and a survey of ChIP-seq analysis tools, discuss experimental design, and describe a detailed ChIP-seq method.

AURKA overexpression accompanies dysregulation of DNA-damage response genes in invasive urothelial cell carcinoma

Invasive urothelial cell carcinoma (UCC) is characterized by increased chromosomal instability and follows an aggressive clinical course in contrast to non-invasive disease. To identify molecular processes that confer and maintain an aggressive malignant phenotype, we used a high-throughput genome-wide approach to interrogate a cohort of high and low clinical risk UCC tumors. Differential expression analyses highlighted cohesive dysregulation of critical genes involved in the G(2)/M checkpoint in aggressive UCC. Hierarchical clustering based on DNA Damage Response (DDR) genes separated tumors according to a pre-defined clinical risk phenotype. Using array-comparative genomic hybridization, we confirmed that the DDR was disrupted in tumors displaying high genomic instability. We identified DNA copy number gains at 20q13.2-q13.3 (AURKA locus) and determined that overexpression of AURKA accompanied dysregulation of DDR genes in high risk tumors. We postulated that DDR-deficient UCC tumors are advantaged by a selective pressure for AURKA associated override of M phase barriers and confirmed this in an independent tissue microarray series. This mechanism that enables cancer cells to maintain an aggressive phenotype forms a rationale for targeting AURKA as a therapeutic strategy in advanced stage UCC.

New androgen receptor genomic targets show an interaction with the ETS1 transcription factor

The androgen receptor (AR) initiates important developmental and oncogenic transcriptional pathways. The AR is known to bind as a homodimer to 15-base pair bipartite palindromic androgen-response elements; however, few direct AR gene targets are known. To identify AR promoter targets, we used chromatin immunoprecipitation with on-chip detection of genomic fragments. We identified 1,532 potential AR-binding sites, including previously known AR gene targets. Many of the new AR target genes show altered expression in prostate cancer. Analysis of sequences underlying AR-binding sites showed that more than 50% of AR-binding sites did not contain the established 15 bp AR-binding element. Unbiased sequence analysis showed 6-bp motifs, which were significantly enriched and were bound directly by the AR in vitro. Binding sequences for the avian erythroblastosis virus E26 homologue (ETS) transcription factor family were also highly enriched, and we uncovered an interaction between the AR and ETS1 at a subset of AR promoter targets.

Phylogenetic insights of Avena genus based on genomic analysis of 45S rDNA molecular organization

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2014

Penicillium glabrum cork colonising isolates – preliminary analysis of their genomic similari

Cork stopper manufacturing process includes an operation, known as stabilisation, by which humid cork slabs are extensively colonised by fungi. The effects of fungal growth on cork are yet to be completely understood and are considered to be involved in the so called “cork taint” of bottled wine. It is essential to identify environmental constraints which define the appearance of the colonising fungal species and to trace their origin to the forest and/or as residents in the manufacturing space. The present article correlates two sets of data, from consecutive years and the same season, of systematic biologic sampling of two manufacturing units, located in the North and South of Portugal. Chrysonilia sitophila dominance was identified, followed by a high diversity of Penicillium species. Penicillium glabrum, found in all samples, was the most frequent isolated species. P. glabrum intra-species variability was investigated using DNA fingerprinting techniques revealing highly discriminative polymorphic markers in the genome. Cluster analysis of P. glabrum data was discussed in relation to the geographical location of strains, and results suggest that P. glabrum arise from predominantly the manufacturing space, although cork resident fungi can also contrib

Soft tissue sarcomas with complex genomic profiles.

Soft tissue sarcomas (STS) with complex genomic profiles (50% of all STS) are predominantly composed of spindle cell/pleomorphic sarcomas, including leiomyosarcoma, myxofibrosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, malignant peripheral nerve sheath tumor, angiosarcoma, extraskeletal osteosarcoma, and spindle cell/pleomorphic unclassified sarcoma (previously called spindle cell/pleomorphic malignant fibrous histiocytoma). These neoplasms show, characteristically, gains and losses of numerous chromosomes or chromosome regions, as well as amplifications. Many of them share recurrent aberrations (e.g., gain of 5p13-p15) that seem to play a significant role in tumor progression and/or metastatic dissemination. In this paper, we review the cytogenetic, molecular genetic, and clinicopathologic characteristics of the most common STS displaying complex genomic profiles. Features of diagnostic or prognostic relevance will be discussed when needed.

Genomic, Epigenomic and Transcriptomic Molecular Characterization of Splenic Marginal Zone Lymphoma Reveals Recurrent Abnormalities in miR-182

Splenic marginal zone lymphoma (SMZL) is a low grade B-cell non-Hodgkin's lymphoma. The molecular pathology of this entity remains poorly understood. To characterise this lymphoma at the molecular level, we performed an integrated analysis of 1) genome wide genetic copy number alterations 2) gene expression profiles and 3) epigenetic DNA methylation profiles.We have previously shown that SMZL is characterised by recurrent alterations of chromosomes 7q, 6q, 3q, 9q and 18; however, gene resolution oligonucleotide array comparative genomic hybridisation did not reveal evidence of cryptic amplification or deletion in these regions. The most frequently lost 7q32 region contains a cluster of miRNAs. qRT-PCR revealed that three of these (miR-182/96/183) show underexpression in SMZL, and miR-182 is somatically mutated in >20% of cases of SMZL, as well as in >20% of cases of follicular lymphoma, and between 5-15% of cases of chronic lymphocytic leukaemia, MALT-lymphoma and hairy cell leukaemia. We conclude that miR-182 is a strong candidate novel tumour suppressor miRNA in lymphoma.The overall gene expression signature of SMZL was found to be strongly distinct fromthose of other lymphomas. Functional analysis of gene expression data revealed SMZL to be characterised by abnormalities in B-cell receptor signalling (especially through the CD19/21-PI3K/AKT pathway) and apoptotic pathways. In addition, genes involved in the response to viral infection appeared upregulated. SMZL shows a unique epigenetic profile, but analysis of differentially methylated genes showed few with methylation related transcriptional deregulation, suggesting that DNA methylation abnormalities are not a critical component of the SMZL malignant phenotype.

Simple method for detection of MYH11 DNA rearrangements in patients with inv(16)(p13q22) and acute myeloid leukemia.

The pericentric inversion on chromosome 16 [inv(16)(p13q22)] and related t(16;16)(p13;q22) are recurrent aberrations associated with acute myeloid leukemia (AML) M4 Eo. Both abberations result in a fusion of the core binding factor beta (CBFB) and smooth muscle myosin heavy chain gene (MYH11). A selected genomic 6.9-kb BamHl probe detects MYH11 DNA rearrangements in 18 of 19 inv(16)/t(16;16) patients tested using HindIII digested DNA. The rearranged fragments were not detectable after remission in two cases tested, while they were present after relapse in one of these two cases tested.

Cloning of actin genes from a genomic library of the newt, Notophthalmus viridescens

The regenerating urodele limb is a useful model system in which to study, in vivo, the controls of cell proliferation and differentiation. Techniques are available which enable one to experimentally manipulate mitogenic influences upon the blastema, as well the morphogenesis of the regenerating 11mb. Although classical regeneration studies have generated a wealth of knowledge concerning tissue interactions, little 1s known about the process at the level of gene expression. The aim of this project was to clone potentially developmentally regulated genes from a newt genomic library for use in future studies of gene expression during limb regeneration. We decided to clone the cytoskeletal actin gene for the following reasons: 1. its expression reflects the proliferative and differentiatlve states of cells in other systems 2. the high copy number of cytoplasmic actin pseudogenes in other vertebrates and the high degree of evolutionary sequence conservation among actin genes increased the chance of cloning one of the newt cytoplasmic actin genes. 3. Preliminary experiments indicated that a newt actin could probably be identified using an available chick ~-actln gene for a molecular probe. Two independent recombinant phage clones, containing actin homologous inserts, were isolated from a newt genomic library by hybridization with the chick actin probe. Restriction mapping identified actin homologous sequences within the newt DNA inserts which were subcloned into the plasmid pTZ19R. The recombinant plasmids were transformed into the Escherichia coli strain, DHsa. Detailed restriction maps were produced of the 5.7Kb and 3.1Kb newt DNA inserts in the plasmids, designated pTNAl and pTNA2. The short «1.3 Kb) length of the actin homologous sequence in pTNA2 indicated that it was possibly a reverse transcript pseudogene. Problems associated with molecular cloning of DNA sequences from N. viridescens are discussed with respect to the large genome size and abundant highly repetitive DNA sequences.

Improving Short DNA Sequence Alignment with Parallel Computing

Variations in different types of genomes have been found to be responsible for a large degree of physical diversity such as appearance and susceptibility to disease. Identification of genomic variations is difficult and can be facilitated through computational analysis of DNA sequences. Newly available technologies are able to sequence billions of DNA base pairs relatively quickly. These sequences can be used to identify variations within their specific genome but must be mapped to a reference sequence first. In order to align these sequences to a reference sequence, we require mapping algorithms that make use of approximate string matching and string indexing methods. To date, few mapping algorithms have been tailored to handle the massive amounts of output generated by newly available sequencing technologies. In otrder to handle this large amount of data, we modified the popular mapping software BWA to run in parallel using OpenMPI. Parallel BWA matches the efficiency of multithreaded BWA functions while providing efficient parallelism for BWA functions that do not currently support multithreading. Parallel BWA shows significant wall time speedup in comparison to multithreaded BWA on high-performance computing clusters, and will thus facilitate the analysis of genome sequencing data.

The in vivo characterization of the DNA repair gene apn-1 in the model organism Caenorhabditis elegans

Les sites apuriniques/apyrimidinique (AP) représentent une forme de dommage à l’ADN hautement mutagène et ce type de dommage peut survenir spontanément ou être induit par une variété d’agents. Afin de préserver la stabilité génomique, deux familles d’endonucléases de type AP, endo-IV et exo-III, sont nécessaires pour contrecarrer les effets mutagènes des sites AP. Malgré l’identification de membres des deux familles dans plusieurs organismes unicellulaire tels que E.coli et S. cerevisiae, aucun membre de la famille endo-IV n’a été identifié chez les organismes multicellulaires à l’exception de C. elegans et de C. briggsae. Nous avons donc décidé d’investiguer l’importance biologique de APN-1 chez C. elegans par l’utilisation d’une approche de knockdown du gène. Dans notre étude, nous avons montré que le knockdown du gène apn-1 chez C. elegans, en utilisant des ARN d’interférence (ARNi), cause une accumulation de mutations spontanées et induites par des drogues résultant en un délai de l’éclosion des œufs ainsi que par une diminution de la survie et de la longévité des vers adultes. De plus, nous avons montré que cette accumulation de mutations mène à un délai dans la progression du cycle cellulaire durant l’embryogénèse, représentant possiblement une explication du délai dans l’éclosion des œufs. Nous avons montré qu’il y avait une augmentation du niveau de mutations dans la gorge des vers, sans toutefois pouvoir confirmer la distribution de APN-1 qui possède une étiquette GFP. Les animaux transgéniques APN-1-GFP n’exprimaient pas suffisamment de la protéine de fusion pour permettre une visualisation à l’aide d’un microscope à fluorescence, mais la protéine a été détectée par immunobuvardage de type western. Les animaux transgéniques APN-1-GFP étaient instables et avaient des phénotypes concordants avec les défauts génétiques. En conclusion, il semble que C. elegans aie évolué afin de retenir un niveau de base de APN-1 jouant ainsi un rôle versatile afin de maintenir l’intégrité génétique d’autant plus que cet organisme semble manquer plusieurs enzymes de la voie de réparation par excision de base.