Search

Genomic tumor testing (GTT) is an emerging technology aimed at identifying variants in tumors that can be targeted with genomically matched drugs. Due to limited resources, rural patients receiving care in community oncology settings may be less likely to benefit from GTT. We analyzed GTT results and observational clinical outcomes data from patients enrolled in the Maine Cancer Genomics Initiative (MCGI), which provided access to GTTs; clinician educational resources; and genomic tumor boards in community practices in a predominantly rural state. 1603 adult cancer patients completed enrollment; 1258 had at least one potentially actionable variant identified. 206 (16.4%) patients received a total of 240 genome matched treatments, of those treatments, 64% were FDA-approved in the tumor type, 27% FDA-approved in a different tumor type and 9% were given on a clinical trial. Using Inverse Probability of Treatment Weighting to adjust for baseline characteristics, a Cox proportional hazards model demonstrated that patients who received genome matched treatment were 31% less likely to die within 1 year compared to those who did not receive genome matched treatment (HR: 0.69; 95% CI: 0.52–0.90; p-value: 0.006). Overall, GTT through this initiative resulted in levels of genome matched treatment that were similar to other initiatives, however, clinical trials represented a smaller share of treatments than previously reported, and "off-label" treatments represented a greater share. Although this was an observational study, we found evidence for a potential 1-year survival benefit for patients who received genome matched treatments. These findings suggest that when disseminated and implemented with a supportive infrastructure, GTT may benefit cancer patients in rural community oncology settings, with further work remaining on providing genome-matched clinical trials. [ABSTRACT FROM AUTHOR]

PURPOSE: The Maine Cancer Genomics Initiative (MCGI) aimed to overcome patient- and provider-level barriers to using genomic tumor testing (GTT) in rural practices by providing genomic tumor boards (GTBs), clinician education, and access to comprehensive large-panel next-generation sequencing to all patients with cancer in Maine. This paper describes the successful implementation of the initiative and three key services made operative between 2016 and 2020. METHODS: A community-inclusive, hub-and-spoke approach was taken to implement the three program components: (1) a centralized GTB program; (2) a modular online education program, designed using an iterative approach with broad clinical stakeholders; and (3) GTT free of charge to clinicians and patients. Implementation timelines, participation metrics, and survey data were used to describe the rollout. RESULTS: The MCGI was launched over an 18-month period at all 19 oncology practices in the State. Seventy-nine physicians (66 medical oncologists, 5 gynecologic oncologists, 1 neuro-oncologist, and 7 pediatric oncologists) enrolled on the study, representing 100% of all practicing oncologists in Maine. Between July 2017 and September 2020, 1610 patients were enrolled. A total of 515 cases were discussed by 47 (73%) clinicians in 196 GTBs. Clinicians who participated in the GTBs enrolled significantly more patients on the study, stayed in Maine, and reported less time spent in clinical patient care. CONCLUSION: The MCGI was able to engage geographically and culturally disparate cancer care practices in a precision oncology program using a hub-and-spoke model. By facilitating access to GTT, structured education, and GTBs, we narrowed the gap in the implementation of precision oncology in one of the most rural states in the country. MCGI aimed to overcome patient- and provider-level barriers to GTT in rural practices by providing GTBs, clinician education, and access to large-panel NGS to all oncologists and their patients in Maine. [ABSTRACT FROM AUTHOR]

Cancer functional genomics is the study of how genetic, epigenetic, and transcriptional alterations affect cancer phenotypes, such as growth and therapeutic response. Here, we comment on how, taking advantage of next generation sequencing, functional genomics, often combined with systems biology approaches, has revealed novel cancer vulnerabilities beyond the original paradigm of one gene-one phenotype. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) and ovarian carcinomas (OvCas) with BRCA1 promoter methylation (BRCA1meth) respond more poorly to alkylating agents compared to those bearing mutations in BRCA1 and BRCA2 (BRCAmut). This is a conundrum given the biologically equivalent homologous recombination deficiency (HRD) induced by these genetic and epigenetic BRCA perturbations. We dissected this problem through detailed genomic analyses of TNBC and OvCa cohorts and experimentation with patient-derived xenografts and genetically engineered cell lines. We found that despite identical downstream genomic mutational signatures associated with BRCA1meth and BRCAmut states, BRCA1meth uniformly associates with poor outcomes. Exposure of BRCA1meth TNBCs to platinum chemotherapy, either as clinical treatment of a patient or as experimental in vivo exposure of preclinical patient derived xenografts, resulted in allelic loss of BRCA1 methylation and increased BRCA1 expression and platinum resistance. These data suggest that, unlike BRCAmut cancers, where BRCA loss is a genetically "fixed" deficiency state, BRCA1meth cancers are highly adaptive to genotoxin exposure and, through reversal of promoter methylation, recover BRCA1 expression and become resistant to therapy. We further found a specific augmented immune transcriptional signal associated with enhanced response to platinum chemotherapy but only in patients with BRCA-proficient cancers. We showed how integrating both this cancer immune signature and the presence of BRCA mutations results in more accurate predictions of patient response when compared to either HRD status or BRCA status alone. This underscores the importance of defining BRCA heterogeneity in optimizing the predictive precision of assigning response probabilities in TNBC and OvCa. Breaking down BRCA heterogeneity: Loss of BRCA1 or BRCA2 (BRCA) activity is common in triple-negative breast cancer (TNBC) and ovarian cancer (OvCa); however, the type of alteration can result in different treatment responses. Here, Menghi et al. examined the differences in response to platinum therapy of patients with TNBC and OvCa who had BRCA loss or genetic alterations as compared to promotor methylation of BRCA. They saw that both patients and mice with promotor methylation were able to adaptively lose methylation, gain BRCA1 expression, and acquire resistance to platinum therapy. These findings have broad implications in using BRCA alteration status to better predict patient. [ABSTRACT FROM AUTHOR]

Employers have begun to offer voluntary workplace genomic testing (wGT) as part of employee wellness benefit programs, but few empirical studies have examined the ethical, legal, and social implications (ELSI) of wGT. To better understand employee perspectives on wGT, employees were surveyed at a large biomedical research institution. Survey respondents were presented with three hypothetical scenarios for accessing health-related genomic testing: via (1) their doctor; (2) their workplace; and 3) a commercial direct-to-consumer (DTC) genetic testing company. Overall, 594 employees (28%) responded to the survey. Respondents indicated a preference for genomic testing in the workplace setting (70%; 95% CI 66–74%), followed by doctor's office (54%; 95% CI 50–58%), and DTC testing (20%; 95% CI 17–24%). Prior to participating in wGT, respondents wanted to know about confidentiality of test results (79%), existence of relevant laws and policies (70%), and privacy protection (64%). Across scenarios, 92% of respondents preferred to view the test results with a genetic counselor. These preliminary results suggest that many employees are interested and even prefer genetic testing in the workplace and would prefer testing with support from genetic health professionals. Confirmation in more diverse employer settings will be needed to generalize such findings. [ABSTRACT FROM AUTHOR]

Small, approximately 10-kilobase microhomology-mediated tandem duplications are abundant in the genomes of BRCA1-linked but not BRCA2-linked breast cancer. Here we define the mechanism underlying this rearrangement signature. We show that, in primary mammalian cells, BRCA1, but not BRCA2, suppresses the formation of tandem duplications at a site-specific chromosomal replication fork barrier imposed by the binding of Tus proteins to an array of Ter sites. BRCA1 has no equivalent role at chromosomal double-stranded DNA breaks, indicating that tandem duplications form specifically at stalled forks. Tandem duplications in BRCA1 mutant cells arise by a replication restart-bypass mechanism terminated by end joining or by microhomology-mediated template switching, the latter forming complex tandem duplication breakpoints. Solitary DNA ends form directly at Tus-Ter, implicating misrepair of these lesions in tandem duplication formation. Furthermore, BRCA1 inactivation is strongly associated with ~10 kilobase tandem duplications in ovarian cancer. This tandem duplicator phenotype may be a general signature of BRCA1-deficient cancer. [ABSTRACT FROM AUTHOR]

Since the emergence of the pandemic H1N1pdm09 virus in Mexico and California, biannual increases in the number of cases have been detected in Mexico. As observed in previous seasons, pandemic A/H1N1 09 virus was detected in severe cases during the 2011–2012 winter season and finally, during the 2013–2014 winter season it became the most prevalent influenza virus. Molecular and phylogenetic analyses of the whole viral genome are necessary to determine the antigenic and pathogenic characteristics of influenza viruses that cause severe outcomes of the disease. In this paper, we analyzed the evolution, antigenic and genetic drift of Mexican isolates from 2009, at the beginning of the pandemic, to 2014. We found a clear variation of the virus in Mexico from the 2011–2014 season due to different markers and in accordance with previous reports. In this study, we identified 13 novel substitutions with important biological effects, including virulence, T cell epitope presented by MHC and host specificity shift and some others substitutions might have more than one biological function. The systematic monitoring of mutations on whole genome of influenza A pH1N1 (2009) virus circulating at INER in Mexico City might provide valuable information to predict the emergence of new pathogenic influenza virus [ABSTRACT FROM AUTHOR]

The mouse has been a major model system for biomedical research. Gene editing technologies will further increase its importance for studying the human condition and human diseases. [ABSTRACT FROM AUTHOR]

Next-generation sequencing studies have revealed genome-wide structural variation patterns in cancer, such as chromothripsis and chromoplexy, that do not engage a single discernable driver mutation, and whose clinical relevance is unclear. We devised a robust genomic metric able to identify cancers with a chromotype called tandem duplicator phenotype (TDP) characterized by frequent and distributed tandem duplications (TDs). Enriched only in triple-negative breast cancer (TNBC) and in ovarian, endometrial, and liver cancers, TDP tumors conjointly exhibit tumor protein p53 (TP53) mutations, disruption of breast cancer 1 (BRCA1), and increased expression of DNA replication genes pointing at rereplication in a defective checkpoint environment as a plausible causal mechanism. The resultant TDs in TDP augment global oncogene expression and disrupt tumor suppressor genes. Importantly, the TDP strongly correlates with cisplatin sensitivity in both TNBC cell lines and primary patient-derived xenografts. We conclude that the TDP is a common cancer chromotype that coordinately alters oncogene/tumor suppressor expression with potential as a marker for chemotherapeutic response. [ABSTRACT FROM AUTHOR]

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. Recent high-throughput analyses of genomic alterations revealed several driver genes and altered pathways in GC. However, therapeutic applications from genomic data are limited, largely as a result of the lack of druggable molecular targets and preclinical models for drug selection. To identify new therapeutic targets for GC, we performed array comparative genomic hybridization (aCGH) of DNA from 103 patients with GC for copy number alteration (CNA) analysis, and whole-exome sequencing from 55 GCs from the same patients for mutation profiling. Pathway analysis showed recurrent alterations in the Wnt signaling [APC, CTNNB1, and DLC1 (deleted in liver cancer 1)], ErbB signaling (ERBB2, PIK3CA, and KRAS), and p53 signaling/apoptosis [TP53 and BCL2L1 (BCL2-like 1)] pathways. In 18.4% of GC cases (19/103), amplification of the antiapoptotic gene BCL2L1 was observed, and subsequently a BCL2L1 inhibitor was shown to markedly decrease cell viability in BCL2L1 -amplified cell lines and in similarly altered patient-derived GC xenografts, especially when combined with other chemotherapeutic agents. In 10.9% of cases (6/55), mutations in DLC1 were found and were also shown to confer a growth advantage for these cells via activation of Rho-ROCK signaling, rendering these cells more susceptible to a ROCK inhibitor. Taken together, our study implicates BCL2L1 and DLC1 as potential druggable targets for specific subsets of GC cases. [ABSTRACT FROM AUTHOR]

Genome-wide functional analyses require high-resolution genome assembly and annotation. We applied ChIA-PET to analyze gene regulatory networks, including 3D chromosome interactions, underlying thyroid hormone (TH) signaling in the frog Xenopus tropicalis. As the available versions of Xenopus tropicalis assembly and annotation lacked the resolution required for ChIA-PET we improve the genome assembly version 4.1 and annotations using data derived from the paired end tag (PET) sequencing technologies and approaches (e.g., DNA-PET [gPET], RNA-PET etc.). The large insert (~10Kb, ~17Kb) paired end DNA-PET with high throughput NGS sequencing not only significantly improved genome assembly quality, but also strongly reduced genome “fragmentation”, reducing total scaffold numbers by ~60%. Next, RNA-PET technology, designed and developed for the detection of full-length transcripts and fusion mRNA in whole transcriptome studies (ENCODE consortia), was applied to capture the 5' and 3' ends of transcripts. These amendments in assembly and annotation were essential prerequisites for the ChIA-PET analysis of TH transcription regulation. Their application revealed complex regulatory configurations of target genes and the structures of the regulatory networks underlying physiological responses. Our work allowed us to improve the quality of Xenopus tropicalis genomic resources, reaching the standard required for ChIA-PET analysis of transcriptional networks. We consider that the workflow proposed offers useful conceptual and methodological guidance and can readily be applied to other non-conventional models that have low-resolution genome data. [ABSTRACT FROM AUTHOR]

Background Structural mutations (SMs) play a major role in cancer development. In some cancers, such as breast and ovarian, DNA double-strand breaks (DSBs) occur more frequently in transcribed regions, while in other cancer types such as prostate, there is a consistent depletion of breakpoints in transcribed regions. Despite such regularity, little is understood about the mechanisms driving these effects. A few works have suggested that protein binding may be relevant, e.g. in studies of androgen receptor binding and active chromatin in specific cell types. We hypothesized that this behavior might be general, i.e. that correlation between protein-DNA binding (and open chromatin) and breakpoint locations is common across divergent cancers. Results We investigated this hypothesis by comprehensively analyzing the relationship among 457 ENCODE protein binding ChIP-seq experiments, 125 DnaseI and 24 FAIRE experiments, and 14,600 SMs from 8 diverse cancer datasets covering 147 samples. In most cancers, including breast and ovarian, we found enrichment of protein binding and open chromatin in the vicinity of SM breakpoints at distances up to 200 kb. Furthermore, for all cancer types we observed an enhanced enrichment in regions distant from genes when compared to regions proximal to genes, suggesting that the SM-induction mechanism is independent from the bias of DSBs to occur near transcribed regions. We also observed a stronger effect for sites with more than one protein bound. Conclusions Protein binding and open chromatin state are associated with nearby SM breakpoints in many cancer datasets. These observations suggest a consistent mechanism underlying SM locations across different cancers. [ABSTRACT FROM AUTHOR]

Background: Genome-wide comparisons of transcription factor binding sites in different species can be used to evaluate evolutionary constraints that shape gene regulatory circuits and to understand how the interaction between transcription factors shapes their binding landscapes over evolution. Results: We have compared the PPARG binding landscapes in macrophages to investigate the evolutionary impact on PPARG binding diversity in mouse and humans for this important nuclear receptor. Of note, only 5% of the PPARG binding sites were shared between the two species. In contrast, at the gene level, PPARG target genes conserved between both species constitute more than 30% of the target genes regulated by PPARG ligand in human macrophages. Moreover, the majority of all PPARG binding sites (55-60%) in macrophages show co-occupancy of the lineage-specification factor PU.1 in both species. Exploring the evolutionary dynamics of PPARG binding sites, we observed that PU.1 co-binding to PPARG sites appears to be important for possible PPARG ancestral functions such as lipid metabolism. Thus we speculate that PU.1 may have guided utilization of these species-specific PPARG conserved binding sites in macrophages during evolution. Conclusions: We propose a model in which PU.1 sites may have served as "anchor" loci for the formation of new and functionally relevant PPARG binding sites throughout evolution. As PU.1 is an essential factor in macrophage biology, such an evolutionary mechanism would allow for the establishment of relevant PPARG regulatory modules in a PU.1-dependent manner and yet permit for nuanced regulatory changes in individual species. [ABSTRACT FROM AUTHOR]

Introduction: We developed an analytic strategy that correlates gene expression and clinical outcomes as a means to identify novel candidate oncogenes operative in breast cancer. This analysis, followed by functional characterization, resulted in the identification of Jumonji Domain Containing 6 (JMJD6) protein as a novel driver of oncogenic properties in breast cancer. Methods: Through microarray informatics, Cox proportional hazards regression was used to analyze the correlation between gene expression and distant metastasis-free survival (DMFS) of patients in 14 independent breast cancer cohorts. JMJD6 emerged as a top candidate gene robustly associated with poor patient survival. Immunohistochemistry, siRNA-mediated silencing, and forced overexpression of JMJD6 in cell-based assays elucidated molecular mechanisms of JMJD6 action in breast cancer progression and shed light on the clinical breast cancer subtypes relevant to JMJD6 action. Results: JMJD6 was expressed at highest levels in tumors associated with worse outcomes, including ER- and basal-like, Claudin-low, Her2-enriched, and ER+ Luminal B tumors. High nuclear JMJD6 protein was associated with ER negativity, advanced grade, and poor differentiation in tissue microarrays. Separation of ER+/LN- patients that received endocrine monotherapy indicated that JMJD6 is predictive of poor outcome in treatment-specific subgroups. In breast cancer cell lines, loss of JMJD6 consistently resulted in suppressed proliferation but not apoptosis, whereas forced stable overexpression increased growth. In addition, knockdown of JMJD6 in invasive cell lines, such as MDA-MB231, decreased motility and invasion, whereas overexpression in MCF-7 cells slightly promoted motility but did not confer invasive growth. Microarray analysis showed that the most significant transcriptional changes occurred in cellproliferation genes and genes of the TGF-β tumor-suppressor pathway. High proliferation was characterized by constitutively high cyclin E protein levels. The inverse relation of JMJD6 expression with TGF-β2 could be extrapolated to the breast cancer cohorts, suggesting that JMJD6 may affect similar pathways in primary breast cancer. Conclusions: JMJD6 is a novel biomarker of tumor aggressiveness with functional implications in breast cancer growth and migration. [ABSTRACT FROM AUTHOR]

Secretory factors that drive cancer progression are attractive immunotherapeutic targets. We used a whole-genome data-mining approach on multiple cohorts of breast tumours annotated for clinical outcomes to discover such factors. We identified Serine protease inhibitor Kazal-type 1 (SPINK1) to be associated with poor survival in estrogen receptor-positive (ER+) cases. Immunohistochemistry showed that SPINK1 was absent in normal breast, present in early and advanced tumours, and its expression correlated with poor survival in ER+ tumours. In ER− cases, the prognostic effect did not reach statistical significance. Forced expression and/or exposure to recombinant SPINK1 induced invasiveness without affecting cell proliferation. However, down-regulation of SPINK1 resulted in cell death. Further, SPINK1 overexpressing cells were resistant to drug-induced apoptosis due to reduced caspase-3 levels and high expression of Bcl2 and phospho-Bcl2 proteins. Intriguingly, these anti-apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain. Thus, SPINK1 affects multiple aggressive properties in breast cancer: survival, invasiveness and chemoresistance. Because SPINK1 effects are abrogated by neutralizing antibodies, we suggest that SPINK1 is a viable potential therapeutic target in breast cancer. [ABSTRACT FROM AUTHOR]

Introduction: Given the role of estrogen in breast carcinogenesis and the modification of estrogen receptor (ER) activity by its biochemical cofactors, we hypothesize that genetic variation within ER cofactor genes alters cellular response to estrogen exposure and consequently modifies the risk for ER-positive breast cancer. Methods: We genotyped 790 tagging SNPs within 60 ER cofactor genes in 1,257 cases and 1,464 controls from Sweden and in 2,215 cases and 1,265 controls from Finland, and tested their associations with either ER-positive or ER-negative breast cancer. Results: Seven SNPs showed consistent association with ER-positive breast cancer in the two independent samples, and six of them were located within PPARGC1B, encoding an ER co-activator, with the strongest association at rs741581 (odds ratio = 1.41, P = 4.84 × 10-5) that survived Bonferroni correction for multiple testing in the combined ER-positive breast cancer sample (Pcorrected = 0.03). Moreover, we also observed significant synergistic interaction (Pinteraction = 0.008) between the genetic polymorphisms within PPARGC1B and ESR1 in ERpositive breast cancer. By contrast, no consistent association was observed in ER-negative breast cancer. Furthermore, we found that administration of estrogen in the MCF-7 cell line induced PPARGC1B expression and enhanced occupancies of ER and RNA polymerase II within the region of SNP association, suggesting the upregulation of PPARGC1B expression by ESR1 activation. Conclusions: Our study revealed that DNA polymorphisms of PPARGC1B, coding a bona fide ER co-activator, are associated with ER-positive breast cancer risk. The feed-forward transcriptional regulatory loop between PPARGC1B and ESR1 further augments their protein interaction, which provides a plausible mechanistic explanation for the synergistic genetic interaction between PPARGC1B and ESR1 in ER-positive breast cancer. Our study also highlights that biochemically and genomically informed candidate gene studies can enhance the discovery of interactive disease susceptibility genes. [ABSTRACT FROM AUTHOR]

Background: The zebrafish is recognized as a versatile cancer and drug screening model. However, it is not known whether the estrogen-responsive genes and signaling pathways that are involved in estrogen-dependent carcinogenesis and human cancer are operating in zebrafish. In order to determine the potential of zebrafish model for estrogen-related cancer research, we investigated the molecular conservation of estrogen responses operating in both zebrafish and human cancer cell lines. Methods: Microarray experiment was performed on zebrafish exposed to estrogen (17β-estradiol; a classified carcinogen) and an anti-estrogen (ICI 182,780). Zebrafish estrogen-responsive genes sensitive to both estrogen and anti-estrogen were identified and validated using real-time PCR. Human homolog mapping and knowledge-based data mining were performed on zebrafish estrogen responsive genes followed by estrogen receptor binding site analysis and comparative transcriptome analysis with estrogen-responsive human cancer cell lines (MCF7, T47D and Ishikawa). Results: Our transcriptome analysis captured multiple estrogen-responsive genes and signaling pathways that increased cell proliferation, promoted DNA damage and genome instability, and decreased tumor suppressing effects, suggesting a common mechanism for estrogen-induced carcinogenesis. Comparative analysis revealed a core set of conserved estrogen-responsive genes that demonstrate enrichment of estrogen receptor binding sites and cell cycle signaling pathways. Knowledge-based and network analysis led us to propose that the mechanism involving estrogen-activated estrogen receptor mediated down-regulation of human homolog HES1 followed by up-regulation cell cycle-related genes (human homologs E2F4, CDK2, CCNA, CCNB, CCNE), is highly conserved, and this mechanism may involve novel crosstalk with basal AHR. We also identified mitotic roles of polo-like kinase as a conserved signaling pathway with multiple entry points for estrogen regulation. Conclusion: The findings demonstrate the use of zebrafish for characterizing estrogen-like environmental carcinogens and anti-estrogen drug screening. From an evolutionary perspective, our findings suggest that estrogen regulation of cell cycle is perhaps one of the earliest forms of steroidal-receptor controlled cellular processes. Our study provides first evidence of molecular conservation of estrogen-responsiveness between zebrafish and human cancer cell lines, hence demonstrating the potential of zebrafish for estrogen-related cancer research. [ABSTRACT FROM AUTHOR]

Despite the role of the estrogen receptor α (ERα) pathway as a key growth driver for breast cells, the phenotypic consequence of exogenous introduction of ERα into ERα‐negative cells paradoxically has been growth inhibition. We mapped the binding profiles of ERα and its interacting transcription factors (TFs), FOXA1 and GATA3 in MCF‐7 breast carcinoma cells, and observed that these three TFs form a functional enhanceosome that regulates the genes driving core ERα function and cooperatively modulate the transcriptional networks previously ascribed to ERα alone. We demonstrate that these enhanceosome occupied sites are associated with optimal enhancer characteristics with highest p300 co‐activator recruitment, RNA Pol II occupancy, and chromatin opening. Most importantly, we show that the transfection of all three TFs was necessary to reprogramme the ERα‐negative MDA‐MB‐231 and BT‐549 cells to restore the estrogen‐responsive growth resembling estrogen‐treated ERα‐positive MCF‐7 cells. Cumulatively, these results suggest that all the enhanceosome components comprising ERα, FOXA1, and GATA3 are necessary for the full repertoire of cancer‐associated effects of the ERα. [ABSTRACT FROM AUTHOR]

To identify genetic susceptibility loci for nasopharyngeal carcinoma (NPC), a genome-wide association study was performed using 464,328 autosomal SNPs in 1,583 NPC affected individuals (cases) and 1,894 controls of southern Chinese descent. The top 49 SNPs from the genome-wide association study were genotyped in 3,507 cases and 3,063 controls of southern Chinese descent from Guangdong and Guangxi. The seven supportive SNPs were further confirmed by transmission disequilibrium test analysis in 279 trios from Guangdong. We identified three new susceptibility loci, TNFRSF19 on 13q12 (rs9510787, Pcombined = 1.53 × 10−9, odds ratio (OR) = 1.20), MDS1-EVI1 on 3q26 (rs6774494, Pcombined = 1.34 × 10−8, OR = 0.84) and the CDKN2A-CDKN2B gene cluster on 9p21 (rs1412829, Pcombined = 4.84 × 10−7, OR = 0.78). Furthermore, we confirmed the role of HLA by revealing independent associations at rs2860580 (Pcombined = 4.88 × 10−67, OR = 0.58), rs2894207 (Pcombined = 3.42 × 10−33, OR = 0.61) and rs28421666 (Pcombined = 2.49 × 10−18, OR = 0.67). Our findings provide new insights into the pathogenesis of NPC by highlighting the involvement of pathways related to TNFRSF19 and MDS1-EVI1 in addition to HLA molecules. [ABSTRACT FROM AUTHOR]

Despite the central role of estrogen exposure in breast and endometrial cancer development and numerous studies of genes in the estrogen metabolic pathway, polymorphisms within the pathway have not been consistently associated with these cancers. We posit that this is due to the complexity of multiple weak genetic effects within the metabolic pathway that can only be effectively detected through multi-variant analysis. We conducted a comprehensive association analysis of the estrogen metabolic pathway by interrogating 239 tagSNPs within 35 genes of the pathway in three tumor samples. The discovery sample consisted of 1,596 breast cancer cases, 719 endometrial cancer cases, and 1,730 controls from Sweden; and the validation sample included 2,245 breast cancer cases and 1,287 controls from Finland. We performed admixture maximum likelihood (AML)-based global tests to evaluate the cumulative effect from multiple SNPs within the whole metabolic pathway and three sub-pathways for androgen synthesis, androgen-to-estrogen conversion, and estrogen removal. In the discovery sample, although no single polymorphism was significant after correction for multiple testing, the pathway-based AML global test suggested association with both breast (pglobal = 0.034) and endometrial (pglobal = 0.052) cancers. Further testing revealed the association to be focused on polymorphisms within the androgen-to-estrogen conversion sub-pathway, for both breast (pglobal = 0.008) and endometrial cancer (pglobal = 0.014). The sub-pathway association was validated in the Finnish sample of breast cancer (pglobal = 0.015). Further tumor subtype analysis demonstrated that the association of the androgen-to-estrogen conversion sub-pathway was confined to postmenopausal women with sporadic estrogen receptor positive tumors (pglobal = 0.0003). Gene-based AML analysis suggested CYP19A1 and UGT2B4 to be the major players within the sub-pathway. Our study indicates that the composite genetic determinants related to the androgen-estrogen conversion are important for the induction of two hormone-associated cancers, particularly for the hormone-driven breast tumour subtypes. [ABSTRACT FROM AUTHOR]

The article presents questions and answers related to ChIP-seq and gene regulation including the usefulness of DNA binding sites of proteins such as transcription factors, technical problems with ChIP-seq and how peak calling is done.

Background: The pathophysiology of obesity and type 2 diabetes mellitus is associated with abnormalities in endocrine signaling in adipose tissue and one of the key signaling affectors operative in these disorders is the nuclear hormone transcription factor peroxisome proliferator-activated receptor-γ (PPARγ). PPARγ has pleiotropic functions affecting a wide range of fundamental biological processes including the regulation of genes that modulate insulin sensitivity, adipocyte differentiation, inflammation and atherosclerosis. To date, only a limited number of direct targets for PPARγ have been identified through research using the well established pre-adipogenic cell line, 3T3-L1. In order to obtain a genome-wide view of PPARγ binding sites, we applied the pair end-tagging technology (ChIP-PET) to map PPARγ binding sites in 3T3-L1 preadipocyte cells. Methodology/Principal Findings: Coupling gene expression profile analysis with ChIP-PET, we identified in a genome-wide manner over 7700 DNA binding sites of the transcription factor PPARγ and its heterodimeric partner RXR during the course of adipocyte differentiation. Our validation studies prove that the identified sites are bona fide binding sites for both PPARc and RXR and that they are functionally capable of driving PPARγ specific transcription. Our results strongly indicate that PPARγ is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation. Additionally, we find that PPARγ/RXR association is enriched within the proximity of the 5′ region of the transcription start site and this association is significantly associated with transcriptional up-regulation of genes involved in fatty acid and lipid metabolism confirming the role of PPARγ as the master transcriptional regulator of adipogenesis. Evolutionary conservation analysis of these binding sites is greater when adjacent to up-regulated genes than down-regulated genes, suggesting the primordial function of PPARγ/RXR is in the induction of genes. Our functional validations resulted in identifying novel PPARγ direct targets that have not been previously reported to promote adipogenic differentiation. Conclusions/Significance: We have identified in a genome-wide manner the binding sites of PPARγ and RXR during the course of adipogenic differentiation in 3T3L1 cells, and provide an important resource for the study of PPARc function in the context of adipocyte differentiation. [ABSTRACT FROM AUTHOR]

The precision of genome sequences, together with advanced computational approaches, allows complex, clinically relevant biological systems to be examined. Here, I describe the experiences of the Genome Institute of Singapore (GIS) in using genome-to-systems strategies to accelerate biomedical research. To maximize clinically relevant output, we have explored an organizational strategy that encourages coordinated experimentation among investigators with diverse skills and interests through building a culture of integrative science. Our experience suggests that systems biomedicine is a real and potentially fruitful strategy for translational research. [ABSTRACT FROM AUTHOR]

The ability to perform large-scale, expression-based chemogenomics on whole adult organisms, as in invertebrate models (worm and fly), is highly desirable for a vertebrate model but its feasibility and potential has not been demonstrated. We performed expression-based chemogenomics on the whole adult organism of a vertebrate model, the zebrafish, and demonstrated its potential for large-scale predictive and discovery chemical biology. Focusing on two classes of compounds with wide implications to human health, polycyclic (halogenated) aromatic hydrocarbons [P(H)AHs] and estrogenic compounds (ECs), we generated robust prediction models that can discriminate compounds of the same class from those of different classes in two large independent experiments. The robust expression signatures led to the identification of biomarkers for potent aryl hydrocarbon receptor (AHR) and estrogen receptor (ER) agonists, respectively, and were validated in multiple targeted tissues. Knowledge-based data mining of human homologs of zebrafish genes revealed highly conserved chemical-induced biological responses/effects, health risks, and novel biological insights associated with AHR and ER that could be inferred to humans. Thus, our study presents an effective, high-throughput strategy of capturing molecular snapshots of chemical-induced biological states of a whole adult vertebrate that provides information on biomarkers of effects, deregulated signaling pathways, and possible affected biological functions, perturbed physiological systems, and increased health risks. These findings place zebrafish in a strategic position to bridge the wide gap between cell-based and rodent models in chemogenomics research and applications, especially in preclinical drug discovery and toxicology. [ABSTRACT FROM AUTHOR]

Protocadherins (PCDH), localized to synaptic junctions, contribute to the formation of neuronal networks during brain development; thus, it is speculated that protocadherins may play a role in evolution of neuronal complexity. While protocadherin genes are highly conserved in vertebrates, EST evidence from the locus suggests apparently species-specific cis-antisense transcripts. Novel cis-antisense transcripts, which partially overlap the PCDHα12 variable exon, PCDHβ3 single-exon gene, and PCDHψ5 unprocessed pseudogene in the human 5q31 PCDHα/β/γ gene cluster and which are coexpressed with sense-strand transcripts in fetal and adult brain, were identified computationally and validated by gene-specific strand-specific reverse transcriptase PCR (SSRTPCR) and sequencing. Absence of antisense transcripts arising from equivalent genomic locations in mouse indicates that the antisense transcripts originated in the primates after the primate-rodent divergence. Furthermore, not all expected orthologues of human sense and antisense PCDH transcripts were detected in rhesus macaque brain, implying that protocadherin expression patterns differ between primate species. RT followed by quantitative real-time PCR (QPCR) analysis of the three genes in the brain of all three species, and of the PCDHβ15 gene paralogous to PCDHψ5 in human and rhesus, revealed that the presence of antisense transcripts was significantly associated with lower sense expression levels across all orthologues. This inverse relationship, along with the pattern of sense and antisense coexpression in the brain, is consistent with a regulatory role for the primate-specific PCDH cis-antisense transcripts, which may represent recent evolutionary inventions modulating the activity of this conserved gene cluster. [ABSTRACT FROM AUTHOR]

Background: Postmenopausal hormone-replacement therapy (HRT) increases breast-cancer risk. The influence of HRT on the biology of the primary tumor, however, is not well understood. Methods: We obtained breast-cancer gene expression profiles using Affymetrix human genome U133A arrays. We examined the relationship between HRT-regulated gene profiles, tumor characteristics, and recurrence-free survival in 72 postmenopausal women. Results: HRT use in patients with estrogen receptor (ER) protein positive tumors (n = 72) was associated with an altered regulation of 276 genes. Expression profiles based on these genes clustered ER-positive tumors into two molecular subclasses, one of which was associated with HRT use and had significantly better recurrence free survival despite lower ER levels. A comparison with external data suggested that gene regulation in tumors associated with HRT was negatively correlated with gene regulation induced by short-term estrogen exposure, but positively correlated with the effect of tamoxifen. Conclusion: Our findings suggest that post-menopausal HRT use is associated with a distinct gene expression profile related to better recurrence-free survival and lower ER protein levels. Tentatively, HRT-associated gene expression in tumors resembles the effect of tamoxifen exposure on MCF-7 cells. [ABSTRACT FROM AUTHOR]

The human gut is known to be a reservoir of a wide variety of microbes, including viruses. Many RNA viruses are known to be associated with gastroenteritis; however, the enteric RNA viral community present in healthy humans has not been described. Here, we present a comparative metagenomic analysis of the RNA viruses found in three fecal samples from two healthy human individuals. For this study, uncultured viruses were concentrated by tangential flow filtration, and viral RNA was extracted and cloned into shotgun viral cDNA libraries for sequencing analysis. The vast majority of the 36,769 viral sequences obtained were similar to plant pathogenic RNA viruses. The most abundant fecal virus in this study was pepper mild mottle virus (PMMV), which was found in high concentrations—up to 109 virions per gram of dry weight fecal matter. PMMV was also detected in 12 (66.7%) of 18 fecal samples collected from healthy individuals on two continents, indicating that this plant virus is prevalent in the human population. A number of pepper-based foods tested positive for PMMV, suggesting dietary origins for this virus. Intriguingly, the fecal PMMV was infectious to host plants, suggesting that humans might act as a vehicle for the dissemination of certain plant viruses. [ABSTRACT FROM AUTHOR]

The human gut is known to be a reservoir of a wide variety of microbes, including viruses. Many RNA viruses are known to be associated with gastroenteritis; however, the enteric RNA viral community present in healthy humans has not been described. Here, we present a comparative metagenomic analysis of the RNA viruses found in three fecal samples from two healthy human individuals. For this study, uncultured viruses were concentrated by tangential flow filtration, and viral RNA was extracted and cloned into shotgun viral cDNA libraries for sequencing analysis. The vast majority of the 36,769 viral sequences obtained were similar to plant pathogenic RNA viruses. The most abundant fecal virus in this study was pepper mild mottle virus (PMMV), which was found in high concentrations—up to 109 virions per gram of dry weight fecal matter. PMMV was also detected in 12 (66.7%) of 18 fecal samples collected from healthy individuals on two continents, indicating that this plant virus is prevalent in the human population. A number of pepper-based foods tested positive for PMMV, suggesting dietary origins for this virus. Intriguingly, the fecal PMMV was infectious to host plants, suggesting that humans might act as a vehicle for the dissemination of certain plant viruses. A comparative metagenomic analysis of RNA viruses in the human gut identifies the vast majority as plant pathogens. [ABSTRACT FROM AUTHOR]

Inhibitors of histone deacetylases (HDACIs) are a new generation of anticancer agents that selectively kill tumor cells. However, the molecular basis for their tumor selectivity is not well understood. We investigated the effects of HDACIs on the oncogenic Rb-E2F1 pathway, which is frequently deregulated in human cancers. Here, we report that cancer cells with elevated E2F1 activity, caused either by enforced E2F1 expression, or by E1A oncogene expression, are highly susceptible to HDACI-induced cell death. This E2F1-mediated apoptosis is neither p53- nor p73-dependent but proceeds through selective induction of proapoptotic 8H3-only protein Bim. We show that Bim is a direct target of E2F1 and that HDAC inhibition promotes the recruitment of E2F1 to the Bim promoter. Moreover, silencing of Bim by specific small interfering RNA (siRNA) effectively abolishes the E2F1-mediated cell death sensitization to HDACIs. These findings suggest that the oncogenic E2F1 pathway participates in HDACIs-induced apoptosis in cancer cells and underscore the importance of Bim as a key mediator of oncogene-induced apoptosis. Our study provides an important insight into the molecular mechanism of tumor selectivity of HDACIs and predicts that, clinically, HDACIs will be more effective in tumors with high E2F1 activity. [ABSTRACT FROM AUTHOR]

Perturbations of the p53 pathway are associated with more aggressive and therapeutically refractory tumors. However, molecular assessment of p53 status, by using sequence analysis and immunohistochemistry, are incomplete assessors of p53 functional effects. We posited that the transcriptional fingerprint is a more definitive downstream indicator of p53 function. Herein, we analyzed transcript profiles of 251 p53-sequenced primary breast tumors and identified a clinically embedded 32-gene expression signature that distinguishes p53-mutant and wild-type tumors of different histologies and outperforms sequence-based assessments of p53 in predicting prognosis and therapeutic response. Moreover, the p53 signature identified a subset of aggressive tumors absent of sequence mutations in p53 yet exhibiting expression characteristics consistent with p53 deficiency because of attenuated p53 transcript levels. Our results show the primary importance of p53 functional status in predicting clinical breast cancer behavior. [ABSTRACT FROM AUTHOR]

Zebrafish (Danio rerio) is a well-recognized model for the study of vertebrate developmental genetics, yet at the same time little is known about the transcriptional events that underlie zebrafish embryogenesis. Here we have employed microarray analysis to study the temporal activity of developmentally regulated genes during zebrafish embryogenesis. Transcriptome analysis at 12 different embryonic time points covering five different developmental stages (maternal, blastula, gastrula, segmentation, and pharyngula) revealed a highly dynamic transcriptional profile. Hierarchical clustering, stage-specific clustering, and algorithms to detect onset and peak of gene expression revealed clearly demarcated transcript clusters with maximum gene activity at distinct developmental stages as well as co-regulated expression of gene groups involved in dedicated functions such as organogenesis. Our study also revealed a previously unidentified cohort of genes that are transcribed prior to the mid-blastula transition, a time point earlier than when the zygotic genome was traditionally thought to become active. Here we provide, for the first time to our knowledge, a comprehensive list of developmentally regulated zebrafish genes and their expression profiles during embryogenesis, including novel information on the temporal expression of several thousand previously uncharacterized genes. The expression data generated from this study are accessible to all interested scientists from our institute resource database (http://giscompute.gis.a-star.edu.sg/~govind/zebrafish/data_download.html). [ABSTRACT FROM AUTHOR]

Valproate (VPA) and trichostatin A (TSA), inhibitors of zinc-dependent deacetylase activity, induce reduction in the levels of mRNA encoding oestrogen receptor-α (ERα), resulting in subsequent clearance of ERα protein from breast and ovarian cell lines. Inhibition of oestrogen signalling may account for the endocrine disorders, menstrual abnormalities, osteoporosis and weight gain that occur in a proportion of women treated with VPA for epilepsy or for bipolar mood disorder. Transcriptome profiling revealed that VPA and TSA also modulate the expression of, among others, key regulatory components of the cell cycle. Meta-analysis of genes directly responsive to oestrogen indicates that VPA and TSA have a generally antioestrogenic profile in ERα positive cells. Concomitant treatment with cycloheximide prevented most of these changes in gene expression, including downregulation of ERα mRNA, indicating that a limited number of genes signal a hyperacetylated state within cells. Three members of the NAD-dependent deacetylases, the sirtuins, are upregulated by VPA and by TSA and sirtuin activity contributes to loss of ERα expression. However, prolonged inhibition of the sirtuins by sirtinol also induces loss of ERα from cells. Mechanistically, we show that VPA invokes reversible promoter shutoff of the ERα, pS2 and cyclin D1 promoters, by inducing recruitment of methyl cytosine binding protein 2 (MeCP2) with concomitant exclusion of the maintenance methylase DNMT1. Furthermore, we demonstrate that, in the presence of VPA, local DNA methylation, deacetylation and demethylation of activated histones and recruitment of inhibitory complexes occurs on the pS2 promoter.Oncogene (2005) 24, 4894–4907. doi:10.1038/sj.onc.1208662; published online 2 May 2005 [ABSTRACT FROM AUTHOR]

Microarray technology is a high-throughput platform for the comprehensive analysis of gene expression on a whole genome scale. Most approaches use a solid phase surface to geographically fix specific gene probes and quantification is based on radiometric, fluorometric, or electrochemical detection of hybridized biomolecules such as RNA, DNA, or protein. The technique produces two-dimensional "images" of gene expression and has been discussed in the context of in vitro "molecular imaging." The major challenge in using this technology is the analysis of its massive "image" data output which requires computational means for interpretation, a heightened need for quality data, and precise experimental design. We will discuss the current technologies focused on nucleic-acid-based microarray platforms. This technology has been extended to the detection of DNA binding complexes, single nucleotide polymorphisms, and in DNA copy number assessment with reasonable success. The future focus will be on improving the probe density, array flexibility, and the dynamic range of detection of the technical platform. On a larger scale, data integration and visualization, and on the intelligent utilization of prior biologic information will be the computational challenge. [ABSTRACT FROM AUTHOR]

Activating mutations of KIT and platelet-derived growth factor receptora(PDGFRA) are known to be alternative and mutually exclusive genetic events in the development of gastrointestinal stromal tumors (GISTs). We examined the effect of the mutations of these two genes on the gene expression profile of 22 GISTs using the oligonucleotide microarray. Mutations of KIT and PDGFRA were found in 17 cases and three cases, respectively. The remaining two cases had no detectable mutations in either gene. The mutation status of KIT and PDGFRA was directly related to the expression levels of activated KIT and PDGFRA, and was also related to the different expression levels of activated proteins that play key roles in the downstream of the receptor tyrosine kinase III family. To evaluate the impact of mutation status and the importance of the type of mutation in gene expression and clinical features, microarray-derived data from 22 GISTs were interpreted using a principal component analysis (PCA). Three relevant principal component representing mutation of KIT, PDGFRA and chromosome 14q deletion were identified from the interpretation of the oligonucleotide microarray data with PCA. After supervised analysis, there was at least a two fold difference in expression between GISTs with KIT and PDGFRA mutation in 70 genes. Our findings demonstrate that mutations of KIT and PDGFRA affect differential activation and expression of some genes, and can be used for the molecular classification of GISTs.Oncogene (2005) 24, 1066-1074. doi:10.1038/sj.onc.1208358 Published online 27 December 2004 [ABSTRACT FROM AUTHOR]

Background Epidemiological investigations of infectious disease are mainly dependent on indirect contact information and only occasionally assisted by characterization of pathogen sequence variation from clinical isolates. Direct sequence analysis of the pathogen, particularly at a population level, is generally thought to be too cumbersome, technically difficult, and expensive. We present here a novel application of mass spectrometry (MS)--based technology in characterizing viral sequence variations that overcomes these problems, and we apply it retrospectively to the severe acute respiratory syndrome (SARS) outbreak in Singapore. Methods and Findings The success rate of the MS-based analysis for detecting SARS coronavirus (SARS-CoV) sequence variations was determined to be 95% with 75 copies of viral RNA per reaction, which is sufficient to directly analyze both clinical and cultured samples. Analysis of 13 SARS-CoV isolates from the different stages of the Singapore outbreak identified nine sequence variations that could define the molecular relationship between them and pointed to a new, previously unidentified, primary route of introduction of SARS-CoV into the Singapore population. Our direct determination of viral sequence variation from a clinical sample also clarified an unresolved epidemiological link regarding the acquisition of SARS in a German patient. We were also able to detect heterogeneous viral sequences in primary lung tissues, suggesting a possible coevolution of quasispecies of virus within a single host. Conclusion This study has further demonstrated the importance of improving clinical and epidemio-logical studies of pathogen transmission through the use of genetic analysis and has revealed the MS-based analysis to be a sensitive and accurate method for characterizing SARS-CoV genetic variations in clinical samples. We suggest that this approach should be used routinely during outbreaks of a wide variety of agents, in order to allow the ... [ABSTRACT FROM AUTHOR]

We have developed a DNA tag sequencing and mapping strategy called gene identification signature (GIS) analysis, in which 5′ and 3′ signatures of full-length cDNAs are accurately extracted into paired-end ditags (PETs) that are concatenated for efficient sequencing and mapped to genome sequences to demarcate the transcription boundaries of every gene. GIS analysis is potentially 30-fold more efficient than standard cDNA sequencing approaches for transcriptome characterization. We demonstrated this approach with 116,252 PET sequences derived from mouse embryonic stem cells. Initial analysis of this dataset identified hundreds of previously uncharacterized transcripts, including alternative transcripts of known genes. We also uncovered several intergenically spliced and unusual fusion transcripts, one of which was confirmed as a trans-splicing event and was differentially expressed. The concept of paired-end ditagging described here for transcriptome analysis can also be applied to whole-genome analysis of cis-regulatory and other DNA elements and represents an important technological advance for genome annotation. [ABSTRACT FROM AUTHOR]

Expression genomics can be defined as the study of the dynamic transciptome and its regulatory elements. Technologies are available that can assess transcripts on a genome-wide scale over time and across many samples. This comprehensive and dynamic database is being used to decipher signalling pathways and to identify new biomarkers and targets. Biomarkers emerging from these studies have prognostic potential and can be used to predict therapeutic outcome. The multiplex nature of this approach not only telescopes the time to discovery, but also allows for detection of complex interactions. Taken together, these capabilities, if carefully used, can speed drug development, enhance the identification of potent drug combinations and identify patient populations that will benefit from these new drugs. [ABSTRACT FROM AUTHOR]