Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Indication of Electromagnetic Field Exposure via RBF-SVM Using Time-Series Features of Zebrafish Locomotion

This paper introduces a novel model based on support vector machine with radial basis function kernel (RBF-SVM) using time-series features of zebrafish (Danio rerio) locomotion exposed to different electromagnetic fields (EMFs) to indicate the corresponding EMF exposure. A group of 14 adult zebrafish was randomly divided into two groups, 7 in each group; the fish of each group have the novel tank test under a sham or real magnetic exposure of 6.78 MHz and about 1 A/m. Their locomotion in the tests was videotaped to convert into the x, y coordinate time-series of the trajectories for reforming time-series matrices according to different time-series lengths. The time-series features of zebrafish locomotion were calculated by the comparative time-series analyzing framework highly comparative time-series analysis (HCTSA), and a limited number of the time-series features that were most relevant to the EMF exposure conditions were selected using the minimum redundancy maximum relevance (mRMR) algorithm for RBF-SVM classification training. Before this, ambient environmental parameters (AEPs) had little effect on the locomotion performance of zebrafish processed by the empirical method, which had been quantitatively verified by regression using another group of 14 adult zebrafish. The results have demonstrated that the purposed model is capable of accurately indicating different EMF exposures. All classification accuracies can be 100%, and the classification precision of several classifiers based on specific parameters and feature sets with specific dimensions can reach higher than 95%. The speculative reason for this result is that the specified EMF has affected the zebrafish neural aspect, which is then reflected in their behaviors. The outcomes of this study have provided a new indication model for EMF exposures and provided a reference for the investigation of the impact of EMF exposure

Transcriptional regulation and functional implication of the grass carp CITED1 (gcCITED1) in the negative regulation of HIF-1

Hypoxia triggers a broad range of gene responses that are primarily mediated by the transcription factor, hypoxia-inducible factor-1 (HIF-1) that complexes with the transcriptional coactivator CREB-binding protein/p300 (CBP/p300). In mammals, members of the CBP/p300-interacting transactivators with ED-rich tail (CITED) family, such as CITED2 and CITED4, bind CBP/p300 with high affinity and thereby negatively regulate HIF-1 transactivation. In fish, we have previously shown that two CITED3 homologues from the hypoxia-tolerant grass carp (Ctenopharyngodon idellus) are induced by hypoxia/HIF-1 and able to inhibit HIF-1 transactivation. Here we report the identification and functional characterization of the grass carp CITED1 (gcCITED1) protein as a new repressor of HIF-1-mediated transcriptional activity. Expression of gcCITED1 mRNA was increased in heart, kidney and liver in vivo after exposure to hypoxia. Luciferase reporter and ChIP assays, respectively, indicated the inducibility of the gcCITED1 promoter by gcHIF-1 and the in vivo binding of gcHIF-1 to the gcCITED1 promoter. Ectopic overexpression of gcCITED1 significantly attenuated HIF-1-dependent transactivation of a HRE-luciferase reporter gene. Furthermore, GST pull-down confirmed that gcCITED1 specifically binds via its CR2 domain to the CH1 region of the grass carp p300 coactivator. Overall, our findings suggest that the hypoxia/gcHIF-1-inducible gcCITED1 may function in a negative feedback loop to regulate gcHIF-1 activity in response to hypoxia stress

Assessment of parental beno[a]pyrene exposure-induced cross-generational neurotoxicity and changes in offspring sperm DNA methylome in medaka fish

Previous studies have revealed that DNA methylation changes could serve as potential genomic markers for environmental benzo[a]pyrene (BaP) exposure and intergenerational inheritance of various physiological impairments (e.g. obesity and reproductive pathologies). As a typical aromatic hydrocarbon pollutant, direct BaP exposure has been shown to induce neurotoxicity. To unravel the inheritance mechanisms of the BaP-induced bone phenotype in freshwater medaka, we conducted whole-genome bisulfte sequencing of F1 sperm and identifed 776 differentially methylated genes (DMGs). Ingenuity pathway analysis revealed that DMGs were signif cantly enriched in pathways associated with neuronal development and function. Therefore, it was hypothesized that parental BaP exposure (1 μg/l, 21 days) causes offspring neurotoxicity. Furthermore, the possibility for sperm methylation as an indicator for a neu rotoxic phenotype was investigated. The F0 adult brains and F1 larvae were analyzed for BaP-induced direct and inherited toxicity. Acetylcholinesterase activity was signifcantly reduced in the larvae, together with decreased swimming velocity. Molecular analysis revealed that the marker genes associated with neuron development and growth (alpha1-tubulin, mbp, syn2a, shh, and gap43) as well as brain development (dlx2, otx2, and krox-20) were universally downregulated in the F1 larvae (3 days post-hatching). While parental BaP exposure at an environmentally relevant concentration could induce neurotoxicity in the developing larvae, the brain function of the exposed F0 adults was unaffected. This indicates that developmental neurotoxicity in larvae may result from impaired neuronal devel opment and differentiation, causing delayed brain growth. The present study demonstrates that the possible adverse health effects of BaP in the environment are more extensive than currently understood. Thus, the possibility of multigenerational BaP toxicity should be included in environmental risk assessments.This work was supported by funding from the Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) (SMSEGL20SC02) and the National Natural Science Foundation of China (No. 41977371). Dr F.S. was supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under award number 1R15ES032936-01

The role of DNA methylation on gene expression in the vertebrae of ancestrally benzo[a]pyrene exposed F1 and F3 male medaka

Benzo[a]pyrene (BaP) is ubiquitously present in the aquatic environment and has been identified as a bone toxicant. Previous studies have demonstrated that ancestral BaP exposure can cause transgenerational bone deformities in fish. Transgenerational effects are thought to be caused by heritable epigenetic changes, such as DNA methylation, histone modification, and non-coding RNAs. To investigate the role of DNA methylation in BaP-induced transgenerational skeletal deformities and the related transcriptomic changes in deformed vertebrae, we examined the vertebrae of male F1 and F3 medaka fish using high-throughput RNA sequencing (RNA-seq) and whole-genome bisulphite sequencing (WGBS). The histological results revealed that osteoblast numbers at the vertebral bone decreased in the BaP-derived F1 and F3 adult males in comparison with the control group. Differentially methylated genes (DMGs) associated with osteoblastogenesis (F1 and F3), chondrogenesis (F1 and F3), and osteoclastogenesis (F3) were identified. However, RNA-seq data did not support the role of DNA methylation in the regulation of genes involved in skeletogenesis since there was very little correlation between the level of differential methylation and gene expression profiles related to skeletogenesis. Although DNA methylation plays a major role in the epigenetic regulation of gene expression, the dysregulation of vertebral gene expression patterns observed in the current study is most likely to be mediated by histone modification and miRNAs. Notably, RNA-seq and WGBS data indicated that genes related to nervous system development are more sensitive to ancestral BaP exposure, indicating a more complex transgenerational phenotype in response to ancestral BaP exposure

Leptin-mediated modulation of steroidogenic gene expression in hypoxic zebrafish embryos: implications for the disruption of sex steroids

Hypoxia can impair reproduction of fishes through the disruption of sex steroids. Here, using zebrafish (Danio rerio) embryos, we investigated (i) whether hypoxia can directly affect steroidogenesis independent of pituitary regulation via modulation of steroidogenic gene expression, and (ii) the role of leptin in hypoxia-induced disruption of steroidogenesis. Exposure of fertilized zebrafish embryos to hypoxia (1.0 mg O₂ L^–1) from 0–72 h postfertilization (hpf), a developmental window when steroidogenesis is unregulated by pituitary influence, resulted in the up-regulation of <i>cyp11a, cyp17</i>, and <i>3β-hsd</i> and the down-regulation of <i>cyp19a</i>. Similar gene expression patterns were observed for embryos exposed to 10 mM cobalt chloride (CoCl₂, a chemical inducer of hypoxia-inducible factor 1, HIF-1), suggesting a regulatory role of HIF-1 in steroidogenesis. Testosterone (T) and estradiol (E2) concentrations in hypoxic embryos were greater and lesser, respectively, relative to the normoxic control, thus leading to an increased T/E2 ratio. Expression of the leptin-a gene (<i>zlep-a</i>) was up-regulated upon both hypoxia and CoCl₂ treatments. Functional assays suggested that under hypoxia, elevated <i>zlep-a</i> expression might activate <i>cyp11a</i> and <i>3β-hsd</i> and inhibit <i>cyp19a</i>. Overall, this study indicates that hypoxia, possibly via HIF-1-induced leptin expression, modulates sex steroid synthesis by acting directly on steroidogenic gene expression

Identification and expression profiling of microRNAs in the brain, liver and gonads of marine medaka (Oryzias melastigma) and in response to hypoxia.

The marine medaka (Oryzias melastigma) has been increasingly used as a fish model for detecting environmental stresses and chemical contaminants in the marine environment. Recent mammalian studies have shown that environmental stresses can alter the expression profiles of microRNAs (miRNAs), leading to transgenerational effects. Here, we use high-throughput Illumina RNA sequencing (RNA-Seq) for miRNA transcriptome analysis of brain, liver, and gonads from sexually mature male and female marine medaka. A total of 128,883,806 filtered sequence reads were generated from six small RNA libraries, identifying a total of 2,125,663 non-redundant sequences. These sequences were aligned and annotated to known animal miRNAs (miRBase) using the BLAST method. A total of 223 distinct miRNA types were identified, with the greatest number expressed in brain tissue. Our data suggested that 55 miRNA types from 34 families are common to all tested tissues, while some of the miRNAs are tissue-enriched or sex-enriched. Quantitative real-time PCR analysis further demonstrated that let-7a, miR-122, and miR-9-3p were downregulated in hypoxic female medaka, while miR-2184 was specifically upregulated in the testis of hypoxic male fish. This is the first study to identify miRNAs in O. melastigma using small RNA deep sequencing technology. Because miRNA expression is highly conserved between marine medaka and other vertebrates, marine medaka may serve as a good model for studies on the functional roles of miRNAs in hypoxia stress response and signaling in marine fish