GCNA Interacts with Spartan and Topoisomerase II to Regulate Genome Stability.

GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan (DVC-1) and Wss1 resolve DNA-protein crosslinks (DPCs), including Topoisomerase-DNA adducts, during DNA replication. Here, we show that GCNA mutants in mouse and C. elegans display defects in genome maintenance including DNA damage, aberrant chromosome condensation, and crossover defects in mouse spermatocytes and spontaneous genomic rearrangements in C. elegans. We show that GCNA and topoisomerase II (TOP2) physically interact in both mice and worms and colocalize on condensed chromosomes during mitosis in C. elegans embryos. Moreover, C. elegans gcna-1 mutants are hypersensitive to TOP2 poison. Together, our findings support a model in which GCNA provides genome maintenance functions in the germline and may do so, in part, by promoting the resolution of TOP2 DPCs.

Whole blood expression profiling from the TREAT trial: insights for the pathogenesis of polyarticular juvenile idiopathic arthritis.

BACKGROUND: The Trial of Early Aggressive Therapy in Juvenile Idiopathic Arthritis (TREAT trial) was accompanied by a once-in-a-generation sample collection for translational research. In this paper, we report the results of whole blood gene expression analyses and genomic data-mining designed to cast light on the immunopathogenesis of polyarticular juvenile idiopathic arthritis (JIA). METHODS: TREAT samples and samples from an independent cohort were analyzed on Affymetrix microarrays and compared to healthy controls. Data from the independent cohort were used to validate the TREAT data. Pathways analysis was used to characterize gene expression profiles. Furthermore, we correlated differential gene expression with new information about functional regulatory elements within the genome to develop models of aberrant gene expression in JIA. RESULTS: There was a strong concordance in gene expression between TREAT samples and the independent cohort. In addition, rheumatoid factor (RF)-positive and RF-negative patients showed only small differences on whole blood expression profiles. Analysis of the combined samples showed 158 genes represented by 176 probes that showed differential expression between TREAT subjects at baseline and healthy controls. None of the differentially expressed genes were encoded within linkage disequilibrium blocks containing single nucleotide polymorphisms known to be associated with risk for JIA. Functional analysis of these genes showed functional associations with multiple processes associated with innate and adaptive immunity, and appeared to reflect overall suppression of STAT1-3/interferon response factor-mediated pathways. CONCLUSIONS: Despite their limitations, whole blood expression profiles clearly distinguish children with polyarticular JIA from healthy controls. Whole blood expression profiles identify several immunologic pathways of biologic relevance that will need to be pursued in homogeneous cell populations in order to clarify mechanisms of pathogenesis. TRIAL REGISTRATION: ClinicalTrials.gov registry #NCT00443430 , originally registered 2 March 2007 and last updated 30 May 2013.

Dynamic tracking of functional gene modules in treated juvenile idiopathic arthritis.

BACKGROUND: We have previously shown that childhood-onset rheumatic diseases show aberrant patterns of gene expression that reflect pathology-associated co-expression networks. In this study, we used novel computational approaches to examine how disease-associated networks are altered in one of the most common rheumatic diseases of childhood, juvenile idiopathic arthritis (JIA). METHODS: Using whole blood gene expression profiles derived from children in a pediatric rheumatology clinical trial, we used a network approach to understanding the impact of therapy and the underlying biology of response/non-response to therapy. RESULTS: We demonstrate that therapy for JIA is associated with extensive re-ordering of gene expression networks, even in children who respond inadequately to therapy. Furthermore, we observe distinct differences in the evolution of specific network properties when we compare children who have been treated successfully with those who have inadequate treatment response. CONCLUSIONS: Despite the inherent noisiness of whole blood gene expression data, our findings demonstrate how therapeutic response might be mapped and understood in pathologically informative cells in a broad range of human inflammatory diseases.

BRD4-targeted therapy induces Myc-independent cytotoxicity in Gnaq/11-mutatant uveal melanoma cells.

Uveal melanoma (UM) is an aggressive intraocular malignancy with limited therapeutic options. Both primary and metastatic UM are characterized by oncogenic mutations in the G-protein alpha subunit q and 11. Furthermore, nearly 40% of UM has amplification of the chromosomal arm 8q and monosomy of chromosome 3, with consequent anomalies of MYC copy number. Chromatin regulators have become attractive targets for cancer therapy. In particular, the bromodomain and extra-terminal (BET) inhibitor JQ1 has shown selective inhibition of c-Myc expression with antiproliferative activity in hematopoietic and solid tumors. Here we provide evidence that JQ1 had cytotoxic activity in UM cell lines carrying Gnaq/11 mutations, while in cells without the mutations had little effects. Using microarray analysis, we identified a large subset of genes modulated by JQ1 involved in the regulation of cell cycle, apoptosis and DNA repair. Further analysis of selected genes determined that the concomitant silencing of Bcl-xL and Rad51 represented the minimal requirement to mimic the apoptotic effects of JQ1 in the mutant cells, independently of c-Myc. In addition, administration of JQ1 to mouse xenograft models of Gnaq-mutant UM resulted in significant inhibition of tumor growth.Collectively, our results define BRD4 targeting as a novel therapeutic intervention against UM with Gnaq/Gna11 mutations.

Increased burden of de novo predicted deleterious variants in complex congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a serious birth defect that accounts for 8% of all major birth anomalies. Approximately 40% of cases occur in association with other anomalies. As sporadic complex CDH likely has a significant impact on reproductive fitness, we hypothesized that de novo variants would account for the etiology in a significant fraction of cases. We performed exome sequencing in 39 CDH trios and compared the frequency of de novo variants with 787 unaffected controls from the Simons Simplex Collection. We found no significant difference in overall frequency of de novo variants between cases and controls. However, among genes that are highly expressed during diaphragm development, there was a significant burden of likely gene disrupting (LGD) and predicted deleterious missense variants in cases (fold enrichment = 3.2, P-value = 0.003), and these genes are more likely to be haploinsufficient (P-value = 0.01) than the ones with benign missense or synonymous de novo variants in cases. After accounting for the frequency of de novo variants in the control population, we estimate that 15% of sporadic complex CDH patients are attributable to de novo LGD or deleterious missense variants. We identified several genes with predicted deleterious de novo variants that fall into common categories of genes related to transcription factors and cell migration that we believe are related to the pathogenesis of CDH. These data provide supportive evidence for novel genes in the pathogenesis of CDH associated with other anomalies and suggest that de novo variants play a significant role in complex CDH cases.

Whole blood gene expression profiling predicts therapeutic response at six months in patients with polyarticular juvenile idiopathic arthritis.

OBJECTIVE: To determine whether gene expression profiles identified in peripheral whole blood samples could be used to determine therapeutic outcome in a cohort of children with newly diagnosed polyarticular juvenile idiopathic arthritis (JIA). METHODS: Whole blood samples from the Trial of Early Aggressive Therapy (TREAT) in JIA patients were analyzed on Illumina microarrays, and differential gene expression was compared to expression in healthy controls. Microarray results were validated by real-time quantitative polymerase chain reaction in an independent cohort of samples. Pathway analysis software was used to characterize gene expression profiles. Support vector machines were used to develop predictive models for different patient classes. RESULTS: Differential gene expression profiles for rheumatoid factor (RF)-positive and RF-negative patients were remarkably similar. Pathway analysis revealed a broad range of affected pathways, consistent with current mechanistic theories. Modeling showed that the prognosis at 6 months was strongly linked to gene expression at presentation, irrespective of treatment. CONCLUSION: Gene expression is linked to therapeutic outcome, and gene expression in the peripheral blood may be a suitable target for a prognostic test.

An information-rich alternative, chemicals testing strategy using a high definition toxicogenomics and zebrafish (Danio rerio) embryos.

Large-scale toxicogenomic screening approaches offer great promise for generating a bias-free system-wide view of toxicological effects and modes-of-action of chemicals and ecotoxicants. However, early applications of microarray technology have identified relatively small groups of responding genes with which to define new targets for analysis by conventional means. We have trialled a more intensive approach to the design and interpretation of array experiments incorporating a balanced interwoven ANOVA design with higher levels of biological replication, a more thorough analysis of errors and false discovery rates, and an analysis of response patterns using gene network models. Zebrafish embryos were exposed from 1.5 h post-fertilization for 72 h to ecotoxicants representing different classes--2,4-dichlorophenol, 3,4-dichloroaniline, pentachlorophenol, and cadmium chloride--at low concentrations producing a developmental disturbance to 10% of embryos and half of this dose. Extracted whole embryo RNA was then analyzed on microarrays. Analysis revealed responses of 3000-5000 genes, which is 10-1000 times greater than previously reported, with significance at lower levels of fold change. Some gene responses were common to multiple toxicants, and others were restricted to just one or two toxicants. The gene expression profiles for the different toxicants were distinctive, and analysis using network-based models provided a high level of detail of affected processes, some of which were novel. This approach provides a more highly refined view of toxic effects, from which meaningful patterns of response can be discerned and related to functional deficits and from which more reliable indicators of toxicological effect can be predicted.