Correction: Whole exome sequencing of high-risk neuroblastoma identifies novel non-synonymous variants.

[This corrects the article DOI: 10.1371/journal.pone.0273280.].

Long Non-Coding RNAs in Hypoxia and Oxidative Stress: Novel Insights Investigating a Piglet Model of Perinatal Asphyxia.

Birth asphyxia is the leading cause of death and disability in young children worldwide. Long non-coding RNAs (lncRNAs) may provide novel targets and intervention strategies due to their regulatory potential, as demonstrated in various diseases and conditions. We investigated cardinal lncRNAs involved in oxidative stress, hypoxia, apoptosis, and DNA damage using a piglet model of perinatal asphyxia. A total of 42 newborn piglets were randomized into 4 study arms: (1) hypoxia-normoxic reoxygenation, (2) hypoxia-3 min of hyperoxic reoxygenation, (3) hypoxia-30 min of hyperoxic reoxygenation, and (4) sham-operated controls. The expression of lncRNAs BDNF-AS, H19, MALAT1, ANRIL, TUG1, and PANDA, together with the related target genes VEGFA, BDNF, TP53, HIF1α, and TNFα, was assessed in the cortex, the hippocampus, the white matter, and the cerebellum using qPCR and Droplet Digital PCR. Exposure to hypoxia-reoxygenation significantly altered the transcription levels of BDNF-AS, H19, MALAT1, and ANRIL. BDNF-AS levels were significantly enhanced after both hypoxia and subsequent hyperoxic reoxygenation, 8% and 100% O2, respectively. Our observations suggest an emerging role for lncRNAs as part of the molecular response to hypoxia-induced damages during perinatal asphyxia. A better understanding of the regulatory properties of BDNF-AS and other lncRNAs may reveal novel targets and intervention strategies in the future.

Assessing nuclear versus mitochondrial cell-free DNA (cfDNA) by qRT-PCR and droplet digital PCR using a piglet model of perinatal asphyxia.

BACKGROUND: Since the discovery more than half a century ago, cell-free DNA (cfDNA) has become an attractive objective in multiple diagnostic, prognostic, and monitoring settings. However, despite the increasing number of cfDNA applications in liquid biopsies, we still lack a comprehensive understanding of the nature of cfDNA including optimal assessment. In the presented study, we continued testing and validation of common techniques for cfDNA extraction and quantification (qRT-PCR or droplet digital PCR) of nuclear- and mitochondrial cfDNA (ncfDNA and mtcfDNA) in blood, using a piglet model of perinatal asphyxia to determine potential temporal and quantitative changes at the levels of cfDNA. METHODS AND RESULTS: Newborn piglets (n = 19) were either exposed to hypoxia (n = 11) or were part of the sham-operated control group (n = 8). Blood samples were collected at baseline (= start) and at the end of hypoxia or at 40-45 min for the sham-operated control group. Applying the qRT-PCR method, ncfDNA concentrations in piglets exposed to hypoxia revealed an increasing trend from 7.1 ng/ml to 9.5 ng/ml for HK2 (hexokinase 2) and from 4.6 ng/ml to 7.9 ng/ml for ß-globulin, respectively, whereas the control animals showed a more balanced profile. Furthermore, median levels of mtcfDNA were much higher in comparison to ncfDNA, but without significant differences between intervention versus the control group. CONCLUSIONS: Both, qRT-PCR and the droplet digital PCR technique identified overall similar patterns for the concentration changes of cfDNA; but, the more sensitive digital PCR methodology might be required to identify minimal responses.

Whole exome sequencing of high-risk neuroblastoma identifies novel non-synonymous variants.

Neuroblastoma (NBL), one of the main death-causing cancers in children, is known for its remarkable genetic heterogeneity and varied patient outcome spanning from spontaneous regression to widespread disease. Specific copy number variations and single gene rearrangements have been proven to be associated with biological behavior and prognosis; however, there is still an unmet need to enlarge the existing armamentarium of prognostic and therapeutic targets. We performed whole exome sequencing (WES) of samples from 18 primary tumors and six relapse samples originating from 18 NBL patients. Our cohort consists of 16 high-risk, one intermediate, and one very low risk patient. The obtained results confirmed known mutational hotspots in ALK and revealed other non-synonymous variants of NBL-related genes (TP53, DMD, ROS, LMO3, PRUNE2, ERBB3, and PHOX2B) and of genes cardinal for other cancers (KRAS, PIK3CA, and FLT3). Beyond, GOSeq analysis determined genes involved in biological adhesion, neurological cell-cell adhesion, JNK cascade, and immune response of cell surface signaling pathways. We were able to identify novel coding variants present in more than one patient in nine biologically relevant genes for NBL, including TMEM14B, TTN, FLG, RHBG, SHROOM3, UTRN, HLA-DRB1, OR6C68, and XIRP2. Our results may provide novel information about genes and signaling pathways relevant for the pathogenesis and clinical course in high-risk NBL.

Quantification of circulating cell-free DNA (cfDNA) in urine using a newborn piglet model of asphyxia.

Cell free DNA (cfDNA) in plasma has been described as a potential diagnostic indicator for a variety of clinical conditions, including neonatal hypoxia. Neonatal hypoxia or perinatal asphyxia is a severe medical condition caused by a temporary interruption in oxygen availability during birth. Previously, we have reported temporal changes of cfDNA detected in blood in a newborn piglet model of perinatal asphyxia. However, cfDNA can also be found in other body liquids, opening for a less invasive diagnostic prospective. The objective of this study was to test and establish a reliable method for the isolation and quantification of cfDNA from urine and to explore changes in the quantities of cfDNA using a newborn piglet model of asphyxia. Animals were exposed to hypoxia-reoxygenation (n = 6), hypoxia-reoxygenation + hypothermia (n = 6) or were part of the sham-operated control group (n = 6) and urine samples (n = 18) were collected at 570 minutes post-intervention. Two alternative applications of cfDNA measurement were tested, an indirect method comprising a centrifugation step together with DNA extraction with magnetic beads versus a direct assessment based on two centrifugation steps. CfDNA concentrations were determined by a fluorescent assay using PicoGreen and by qRT-PCR. Genomic (gDNA) and mitochondrial DNA (mtDNA) cfDNA were determined in parallel, taking into account potential differences in the rates of damages caused by oxidative stress. In contrast to previous publications, our results indicate that the direct method is insufficient. Application of the indirect method obtained with the fluorescence assay revealed mean cfDNA levels (SD) of 1.23 (1.76) ng/ml for the hypoxia samples, 4.47 (6.15) ng/ml for the samples exposed to hypoxia + hypothermia and 2.75 (3.62) ng/ml for the control animals. The mean cfDNA levels in piglets exposed to hypoxia + hypothermia revealed significantly higher cfDNA amounts compared to mean cfDNA levels in the samples purely exposed to hypoxia (p < 0.05); however, no significant difference could be determined when compared to the control group (p = 0.09). Application of the indirect method by qRT-PCR revealed mean cfDNA levels of mtDNA and gDNA at the detection limit of the technique and thus no reliable statistics could be performed between the observed cfDNA levels in the investigated groups. The methodology for detection and monitoring of cfDNA in urine has to be further optimized before it can be applied in a clinical setting in the future.

Temporal patterns of circulating cell-free DNA (cfDNA) in a newborn piglet model of perinatal asphyxia.

Perinatal asphyxia is a severe medical condition resulting from oxygen deficiency (hypoxia) at the time of birth, causing worldwide approximately 680,000 newborn deaths every year. Better prediction of severity of damages including early biomarkers is highly demanded. Elevated levels of circulating cell-free DNA (cfDNA) in blood have been reported for a range of different diseases and conditions, including cancer and prematurity. The objective of this study was to validate methods for assessing cfDNA in blood and cerebrospinal fluid (CSF) and to explore temporal variations in a piglet model of neonatal hypoxia-reoxygenation. Different cfDNA extraction methods in combination with cfDNA detection systems were tested, including a fluorescent assay using SYBR Gold and a qRT-PCR-based technique. Newborn piglets (n = 55) were exposed to hypoxia-reoxygenation, hypoxia-reoxygenation and hypothermia, or were part of the sham-operated control group. Blood was sampled at baseline and at post-intervention, further at 30, 270, and 570 minutes after the end of hypoxia. Applying the fluorescent method, cfDNA concentration in piglets exposed to hypoxia (n = 32) increased from 36.8±27.6 ng/ml prior to hypoxia to a peak level of 61.5±54.9 ng/ml after the intervention and deceased to 32.3±19.1 ng/ml at 570 minutes of reoxygenation, whereas the group of sham-operated control animals (n = 11) revealed a balanced cfDNA profile. Animals exposed to hypoxia and additionally treated with hypothermia (n = 12) expressed a cfDNA concentration of 54.4±16.9 ng/ml at baseline, 39.2±26.9 ng/ml at the end of hypoxia, and of 41.1±34.2 ng/ml at 570 minutes post-intervention. Concentrations of cfDNA in the CSF of piglets exposed to hypoxia revealed at post-intervention higher levels in comparison to the controls. However, these observations were only tendencies and not significant. In a first methodological proof-of-principle study exploring cfDNA using a piglet model of hypoxia-reoxygenation variations in the temporal patterns suggest that cfDNA might be an early indicator for damages caused by perinatal asphyxia.

Identifying in-trans process associated genes in breast cancer by integrated analysis of copy number and expression data.

Genomic copy number alterations are common in cancer. Finding the genes causally implicated in oncogenesis is challenging because the gain or loss of a chromosomal region may affect a few key driver genes and many passengers. Integrative analyses have opened new vistas for addressing this issue. One approach is to identify genes with frequent copy number alterations and corresponding changes in expression. Several methods also analyse effects of transcriptional changes on known pathways. Here, we propose a method that analyses in-cis correlated genes for evidence of in-trans association to biological processes, with no bias towards processes of a particular type or function. The method aims to identify cis-regulated genes for which the expression correlation to other genes provides further evidence of a network-perturbing role in cancer. The proposed unsupervised approach involves a sequence of statistical tests to systematically narrow down the list of relevant genes, based on integrative analysis of copy number and gene expression data. A novel adjustment method handles confounding effects of co-occurring copy number aberrations, potentially a large source of false positives in such studies. Applying the method to whole-genome copy number and expression data from 100 primary breast carcinomas, 6373 genes were identified as commonly aberrant, 578 were highly in-cis correlated, and 56 were in addition associated in-trans to biological processes. Among these in-trans process associated and cis-correlated (iPAC) genes, 28% have previously been reported as breast cancer associated, and 64% as cancer associated. By combining statistical evidence from three separate subanalyses that focus respectively on copy number, gene expression and the combination of the two, the proposed method identifies several known and novel cancer driver candidates. Validation in an independent data set supports the conclusion that the method identifies genes implicated in cancer.