Systematic detection of putative tumor suppressor genes through the combined use of exome and transcriptome sequencing

Abstract Background To identify potential tumor suppressor genes, genome-wide data from exome and transcriptome sequencing were combined to search for genes with loss of heterozygosity and allele-specific expression. The analysis was conducted on the breast cancer cell line HCC1954, and a lymphoblast cell line from the same individual, HCC1954BL. Results By comparing exome sequences from the two cell lines, we identified loss of heterozygosity events at 403 genes in HCC1954 and at one gene in HCC1954BL. The combination of exome and transcriptome sequence data also revealed 86 and 50 genes with allele specific expression events in HCC1954 and HCC1954BL, which comprise 5.4% and 2.6% of genes surveyed, respectively. Many of these genes identified by loss of heterozygosity and allele-specific expression are known or putative tumor suppressor genes, such as BRCA1, MSH3 and SETX, which participate in DNA repair pathways. Conclusions Our results demonstrate that the combined application of high throughput sequencing to exome and allele-specific transcriptome analysis can reveal genes with known tumor suppressor characteristics, and a shortlist of novel candidates for the study of tumor suppressor activities

Occurrence and Treatment of Bone Atrophic Non-Unions Investigated by an Integrative Approach

Recently developed atrophic non-union models are a good representation of the clinical situation in which many nonunions develop. Based on previous experimental studies with these atrophic non-union models, it was hypothesized that in order to obtain successful fracture healing, blood vessels, growth factors, and (proliferative) precursor cells all need to be present in the callus at the same time. This study uses a combined in vivo-in silico approach to investigate these different aspects (vasculature, growth factors, cell proliferation). The mathematical model, initially developed for the study of normal fracture healing, is able to capture essential aspects of the in vivo atrophic non-union model despite a number of deviations that are mainly due to simplifications in the in silico model. The mathematical model is subsequently used to test possible treatment strategies for atrophic non-unions (i.e. cell transplant at post-osteotomy, week 3). Preliminary in vivo experiments corroborate the numerical predictions. Finally, the mathematical model is applied to explain experimental observations and identify potentially crucial steps in the treatments and can thereby be used to optimize experimental and clinical studies in this area. This study demonstrates the potential of the combined in silico-in vivo approach and its clinical implications for the early treatment of patients with problematic fractures

The use of Open Reading frame ESTs (ORESTES) for analysis of the honey bee transcriptome

BACKGROUND: The ongoing efforts to sequence the honey bee genome require additional initiatives to define its transcriptome. Towards this end, we employed the Open Reading frame ESTs (ORESTES) strategy to generate profiles for the life cycle of Apis mellifera workers. RESULTS: Of the 5,021 ORESTES, 35.2% matched with previously deposited Apis ESTs. The analysis of the remaining sequences defined a set of putative orthologs whose majority had their best-match hits with Anopheles and Drosophila genes. CAP3 assembly of the Apis ORESTES with the already existing 15,500 Apis ESTs generated 3,408 contigs. BLASTX comparison of these contigs with protein sets of organisms representing distinct phylogenetic clades revealed a total of 1,629 contigs that Apis mellifera shares with different taxa. Most (41%) represent genes that are in common to all taxa, another 21% are shared between metazoans (Bilateria), and 16% are shared only within the Insecta clade. A set of 23 putative genes presented a best match with human genes, many of which encode factors related to cell signaling/signal transduction. 1,779 contigs (52%) did not match any known sequence. Applying a correction factor deduced from a parallel analysis performed with Drosophila melanogaster ORESTES, we estimate that approximately half of these no-match ESTs contigs (22%) should represent Apis-specific genes. CONCLUSIONS: The versatile and cost-efficient ORESTES approach produced minilibraries for honey bee life cycle stages. Such information on central gene regions contributes to genome annotation and also lends itself to cross-transcriptome comparisons to reveal evolutionary trends in insect genomes

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

Genetic Characterization of Zika Virus Strains: Geographic Expansion of the Asian Lineage

Zika virus (ZIKV) is a mosquito-transmitted flavivirus found in both Africa and Asia. Human infection with the virus may result in a febrile illness similar to dengue fever and many other tropical infections found in these regions. Previously, little was known about the genetic relationships between ZIKV strains collected in Africa and those collected in Asia. In addition, the geographic origins of the strains responsible for the recent outbreak of human disease on Yap Island, Federated States of Micronesia, and a human case of ZIKV infection in Cambodia were unknown. Our results indicate that there are two geographically distinct lineages of ZIKV (African and Asian). The virus has circulated in Southeast Asia for at least the past 50 years, whereupon it was introduced to Yap Island resulting in an epidemic of human disease in 2007, and in 2010 was the cause of a pediatric case of ZIKV infection in Cambodia. This study also highlights the danger of ZIKV introduction into new areas and the potential for future epidemics of human disease

Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury

<p>Abstract</p> <p>Background</p> <p>The sequencing of the <it>D.melanogaster </it>genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of <it>Drosophila </it>protein-coding genes contain one or more alternative exons. A recent transcription map of the <it>Drosophila </it>embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of <it>Drosophila </it>transcriptome.</p> <p>Results</p> <p>Bioinformatic analysis of 1,303 <it>Drosophila </it>ORESTES clusters identified 68 sequences derived from unannotated regions in the current <it>Drosophila </it>genome version (4.3). Of these, a set of 38 was analysed by polyA⁺northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The <it>SP212 </it>gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this <it>locus </it>is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury.</p> <p>Conclusion</p> <p>Using the ORESTES methodology we identified 17 novel exons from low abundance <it>Drosophila </it>transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data.</p

Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data

INTRODUCTION: Diverse microarray and sequencing technologies have been widely used to characterise the molecular changes in malignant epithelial cells in breast cancers. Such gene expression studies to identify markers and targets in tumour cells are, however, compromised by the cellular heterogeneity of solid breast tumours and by the lack of appropriate counterparts representing normal breast epithelial cells. METHODS: Malignant neoplastic epithelial cells from primary breast cancers and luminal and myoepithelial cells isolated from normal human breast tissue were isolated by immunomagnetic separation methods. Pools of RNA from highly enriched preparations of these cell types were subjected to expression profiling using massively parallel signature sequencing (MPSS) and four different genome wide microarray platforms. Functional related transcripts of the differential tumour epithelial transcriptome were used for gene set enrichment analysis to identify enrichment of luminal and myoepithelial type genes. Clinical pathological validation of a small number of genes was performed on tissue microarrays. RESULTS: MPSS identified 6,553 differentially expressed genes between the pool of normal luminal cells and that of primary tumours substantially enriched for epithelial cells, of which 98% were represented and 60% were confirmed by microarray profiling. Significant expression level changes between these two samples detected only by microarray technology were shown by 4,149 transcripts, resulting in a combined differential tumour epithelial transcriptome of 8,051 genes. Microarray gene signatures identified a comprehensive list of 907 and 955 transcripts whose expression differed between luminal epithelial cells and myoepithelial cells, respectively. Functional annotation and gene set enrichment analysis highlighted a group of genes related to skeletal development that were associated with the myoepithelial/basal cells and upregulated in the tumour sample. One of the most highly overexpressed genes in this category, that encoding periostin, was analysed immunohistochemically on breast cancer tissue microarrays and its expression in neoplastic cells correlated with poor outcome in a cohort of poor prognosis estrogen receptor-positive tumours. CONCLUSION: Using highly enriched cell populations in combination with multiplatform gene expression profiling studies, a comprehensive analysis of molecular changes between the normal and malignant breast tissue was established. This study provides a basis for the identification of novel and potentially important targets for diagnosis, prognosis and therapy in breast cancer