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In the event of a large-scale event leading to acute ionizing radiation exposure, high-throughput methods would be required to assess individual dose estimates for triage purposes. Blood-based gene expression is a broad source of biomarkers of radiation exposure which have great potential for providing rapid dose estimates for a large population. Time is a crucial component in radiological emergencies and the shipment of blood samples to relevant laboratories presents a concern. In this study, we performed nanopore sequencing analysis to determine if the technology can be used to detect radiation-inducible genes in human peripheral blood mononuclear cells (PBMCs). The technology offers not only long-read sequencing but also a portable device which can overcome issues involving sample shipment, and provide faster results. For this goal, blood from nine healthy volunteers was 2 Gy ex vivo X irradiated. After PBMC isolation, irradiated samples were incubated along with the controls for 24 h at 37°C. RNA was extracted, poly(A)+ enriched and reverse-transcribed before sequencing. The data generated was analyzed using a Snakemake pipeline modified to handle paired samples. The sequencing analysis identified a radiation signature consisting of 46 differentially expressed genes (DEGs) which included 41 protein-coding genes, a long non-coding RNA and four pseudogenes, five of which have been identified as radiation-responsive transcripts for the first time. The genes in which transcriptional expression is most significantly modified after radiation exposure were APOBEC3H and FDXR, presenting a 25- and 28-fold change on average, respectively. These levels of transcriptional response were comparable to results we obtained by quantitative polymerase chain reaction (qPCR) analysis. In vivo exposure analyses showed a transcriptional radioresponse at 24 h postirradiation for both genes together with a strong dose-dependent response in blood irradiated ex vivo. Finally, extrapolating from the data we obtained, the minimum sequencing time required to detect an irradiated sample using APOBEC3H transcripts would be less than 3 min for a total of 50,000 reads. Future improvements, in sample processing and bioinformatic pipeline for specific radiation-responsive transcript identification, will allow the provision of a portable, rapid, real-time biodosimetry platform based on this new sequencing technology. In summary, our data show that nanopore sequencing can identify radiation-responsive genes and can also be used for identification of new transcripts.

Epidemiological studies have demonstrated an increased leukemia incidence following ionizing radiation exposure, but to date, the target cells and underlying mechanisms of radiation leukemogenesis remain largely unidentified. We engineered a mouse model carrying a different fluorescent marker on each chromosome 2, located inside the minimum deleted region occurring after radiation exposure and recognized as the first leukemogenic event. Using this tailored model, we report that following radiation exposure, more than half of asymptomatic CBA Sfpi1 GFP/mCh mice presented with expanding clones of preleukemic hematopoietic cells harboring a hemizygous interstitial deletion of chromosome 2. Moreover, following isolation of preleukemic hematopoietic stem and progenitor cells irradiated in their native microenvironment, we identified the presence of Sfpi1 point mutations within a subpopulation of these preleukemic cells expanding rapidly (increasing from 6% to 55% in 21 days in peripheral blood in one case), hence identifying for the first time the presence of such cells within a living animal. Importantly, we also report a previously undescribed gender difference in the phenotype of the preleukemic cells and leukemia, suggesting a gender imbalance in the radiation-induced leukemic target cell. In conclusion, we provide novel insights into the sequence of molecular events occurring during the (radiation-induced) leukemic clonal evolution.

This is an Accepted Manuscript of an article published by Taylor & Francis in International Journal of Radiation Biology on 13 Nov 2019, available online: https://www.tandfonline.com/doi/full/10.1080/09553002.2020.1688886. Uploaded in accordance with the publisher's self-archiving policy. NIHR Manchester Biomedical Research Centre

International audience; Though radiotherapy is a local therapy, it has systemic effects mainly influencing immune and inflammation processes. This has important consequences in the long-term prognosis and therapy individualization. Our objective was to investigate immune and inflammation-related changes in the peripheral blood of head and neck cancer patients treated with radiotherapy. Peripheral blood cells, plasma and blood cell-derived RNA were isolated from 23 patients before and at two time points after radiotherapy and cellular immune parameters, plasma protein changes and gene expression alterations were studied. Increased regulatory T cells and increased CTLA4 and PD-1 expression on CD4 cells indicated an immune suppression induced by the malignant condition, which was accentuated by radiotherapy. Circulating dendritic cells were strongly elevated before treatment and were not affected by radiotherapy. Decreased endoglin levels in the plasma of patients before treatment were further decreased by radiotherapy. Expression of the FXDR, SESN1, GADD45, DDB2 and MDM2 radiation-response genes were altered in the peripheral blood cells of patients after radiotherapy. All changes were long-lasting, detectable one month after radiotherapy. In conclusion we demonstrated radiotherapy-induced changes in systemic immune parameters of head and neck cancer patients and proposed markers suitable for patient stratification worth investigating in larger patient cohorts.

International audience; Purpose: The workshop on ‘Individual Radiosensitivity and Radiosusceptibility’ organized by MELODI and CONCERT on Malta in 2018, evaluated the current state of assays to identify sensitive and susceptible subgroups. The authors provide an overview on potential screening assays detecting individuals showing moderate to severe early and late radiation reactions or are at increased risk to develop cancer upon radiation exposure. Conclusion: It is necessary to separate clearly between tissue reactions and stochastic effects such as cancer when comparing the existing literature to validate various test systems. Requirements for the assays are set up. The literature is reviewed for assays that are reliable and robust. Sensitivity and specificity of the assays are regarded and scrutinized for modifying factors. Accuracy of an assay system is required to be more than 90% to balance risks of adverse reactions against risk to fail to cure the cancer. No assay/biomarker is in routine use. Assays that have shown predictive potential for radiosensitivity include SNPs, the RILA assay, and the pATM assay. A tree of risk guideline for radiologists is provided to assist medical treatment decisions. Recommendations for effective research include the setup of common retrospective and prospective cohorts/biobanks to validate current and future tests.

Individual variability in response to radiation exposure is recognised and has often been reported as important in treatment planning. Despite many efforts to identify biomarkers allowing the identification of radiation sensitive patients, it is not yet possible to distinguish them with certainty before the beginning of the radiotherapy treatment. A comprehensive analysis of genome-wide single-nucleotide polymorphisms (SNPs) and a transcriptional response to ionising radiation exposure in twins have the potential to identify such an individual. In the present work, we investigated SNP profile and CDKN1A gene expression in blood T lymphocytes from 130 healthy Caucasians with a complex level of individual kinship (unrelated, mono- or dizygotic twins). It was found that genetic variation accounts for 66% (95% CI 37–82%) of CDKN1A transcriptional response to radiation exposure. We developed a novel integrative multi-kinship strategy allowing investigating the role of genome-wide polymorphisms in transcriptomic radiation response, and it revealed that rs205543 (ETV6 gene), rs2287505 and rs1263612 (KLF7 gene) are significantly associated with CDKN1A expression level. The functional analysis revealed that rs6974232 (RPA3 gene), involved in mismatch repair (p value = 9.68e−04) as well as in RNA repair (p value = 1.4e−03) might have an important role in that process. Two missense polymorphisms with possible deleterious effect in humans were identified: rs1133833 (AKIP1 gene) and rs17362588 (CCDC141 gene). In summary, the data presented here support the validity of this novel integrative data analysis strategy to provide insights into the identification of SNPs potentially influencing radiation sensitivity. Further investigations in radiation response research at the genomic level should be therefore continued to confirm these findings. Electronic supplementary material The online version of this article (10.1007/s10142-019-00658-3) contains supplementary material, which is available to authorized users.

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Studies of the responses of hematopoietic stem and progenitor cells (HSPCs) to low doses of ionizing radiation formed an important aspect of the RISK-IR project ( www.risk-ir.eu ). A brief overview of these studies is presented here. The findings confirm the sensitivity of HSPCs to radiation even at low doses, and illustrate the substantial impact that differentiation state has upon cell sensitivity. The work provides mechanistic support for epidemiological findings of leukemia risk at dose levels used in diagnostic CT imaging, and further suggests that low-dose irradiation may facilitate bone marrow transplantation, a finding that could lead to refinements in clinical practice.

Gene expression (GE) assays have shown great potential for rapid individual radiation dose exposure assessment. The aim of the present study was to optimise GE-based biological dosimetry protocols for radiological emergencies. Experiments were carried out to validate a newly developed protocol (P2) where several steps were optimised and to compare it with the current validated protocol in place in our laboratory (P1). Several donor blood samples from were exposed ex vivo to of the following doses: 0, 0.5, 1, 2 Gy X-rays. Concomitant measurement of transcription level of genes FDXR, P21, PHPT1, CCNG1 and SESN1 plus HPRT (control) was performed. To summarise, both protocols provided similar dose estimates, P1 being completed in 7 hours while P2 in merely 4 hours. Thus, a significant time shortening was achieved leading to a potential increase of throughput capacity. Hence, this new protocol can be recommended for mass radiation casualties triage purposes.

The review aims to discuss the prominence of dietary and metabolic regulators in maintaining hematopoietic stem cell (HSC) function, long-term self-renewal, and differentiation.Most adult stem cells are preserved in a quiescent, nonmotile state in vivo which acts as a "protective state" for stem cells to reduce endogenous stress provoked by DNA replication and cellular respiration as well as exogenous environmental stress. The dynamic balance between quiescence, self-renewal and differentiation is critical for supporting a functional blood system throughout life of an organism. Stress-conditions, for example ionizing radiation exposure can trigger the blood forming HSCs to proliferate and migrate through extramedullary tissues to expand the number of HSCs and increase hematopoiesis. In addition, a wealth of investigation validated that deregulation of this balance plays a critical pathogenic role in various different hematopoietic diseases including the leukemia development.The review summarizes the current knowledge on how alterations in dietary and metabolic factors could alter the risk of leukemia development following ionizing radiation exposure by inhibiting or even reversing the leukemic progression. Understanding the influence of diet, metabolism, and epigenetics on radiation-induced leukemogenesis may lead to the development of practical interventions to reduce the risk in exposed populations.

The RTGene study was focused on the development and validation of new transcriptional biomarkers for prediction of individual radiotherapy patient responses to ionizing radiation. In parallel, for validation purposes, this study incorporated conventional biomarkers of radiation exposure, including the dicentric assay. Peripheral blood samples were taken with ethical approval and informed consent from a total of 20 patients undergoing external beam radiotherapy for breast, lung, gastrointestinal or genitourinary tumors. For the dicentric assay, two samples were taken from each patient: prior to radiotherapy and before the final fraction. Blood samples were set up using standard methods for the dicentric assay. All the baseline samples had dicentric frequencies consistent with the expected background for the normal population. For blood taken before the final fraction, all the samples displayed distributions of aberrations, which are indicative of partial-body exposures. Whole-body and partial-body cytogenetic doses were calculated with reference to a 250-kVp X-ray calibration curve and then compared to the dose to blood derived using two newly developed blood dosimetric models. Initial comparisons indicated that the relationship between these measures of dose appear very promising, with a correlation of 0.88 (P = 0.001). A new Bayesian zero-inflated Poisson finite mixture method was applied to the dicentric data, and partial-body dose estimates showed no significant difference (P > 0.999) from those calculated by the contaminated Poisson technique. The next step will be further development and validation in a larger patient group.

International audience; For triage purposes following a nuclear accident, blood-based gene expression biomarkers can provide rapid dose estimates for a large number of individuals. Ionizing-radiation-responsive genes are regulated through the DNA damage-response pathway, which includes activation of multiple transcription factors. Modulators of this pathway could potentially affect the response of these biomarkers and consequently compromise accurate dose estimation calculations. In the present study, four potential confounding factors were selected: cancer condition, sex, simulated bacterial infection (lipopolysaccharide), and curcumin, an anti-inflammatory/antioxidant agent. Their potential influence on the transcriptional response to radiation of the genes CCNG1 and PHPT1, two biomarkers of radiation exposure ex vivo, was assessed. First, both CCNG1 and PHPT1 were detected in vivo in blood samples from radiotherapy patients and as such were validated as biomarkers of exposure. Importantly, their basal expression level was slightly but significantly affected in vivo by patients' cancer condition. Moreover, lipopolysaccharide stimulation of blood irradiated ex vivo led to a significant modification of CCNG1 and PHPT1 transcriptional response in a dose- and time-dependent manner with opposite regulatory effects. Curcumin also affected CCNG1 and PHPT1 transcriptional response counteracting some of the radiation induction. No differences were observed based on sex. Dose estimations calculated using linear regression were affected by lipopolysaccharide and curcumin. In conclusion, several confounding factors tested in this study can indeed modulate the transcriptional response of CCNG1 and PHPT1 and consequently can affect radiation exposure dose estimations but not to a level which should prevent the biomarkers' use for triage purposes.

International audience; Background: Microsatellite and chromosomal instability have been investigated in Hodgkin lymphoma (HL). Materials and Methods: We studied seven HL cell lines (five Nodular Sclerosis (NS) and two Mixed Cellularity (MC)) and patient peripheral blood lymphocytes (100 NS-HL and 23 MC-HL). Microsatellite instability (MSI) was assessed by PCR. Chromosomal instability and telomere dysfunction were investigated by FISH. DNA repair mechanisms were studied by transcriptomic and molecular approaches. Results: In the cell lines, we observed high MSI in L428 (4/5), KMH2, and HDLM2 (3/5), low MSI in L540, L591, and SUP-HD1, and none in L1236. NS-HL cell lines showed telomere shortening, associated with alterations of nuclear shape. Small cells were characterized by telomere loss and deletion, leading to chromosomal fusion, large nucleoplasmic bridges, and breakage/fusion/bridge (B/F/B) cycles, leading to chromosomal instability. The MC-HL cell lines showed substantial heterogeneity of telomere length. Intrachromosmal double strand breaks induced dicentric chromosome formation, high levels of micronucleus formation, and small nucleoplasmic bridges. B/F/B cycles induced complex chromosomal rearrangements. We observed a similar pattern in circulating lymphocytes of NS-HL and MC-HL patients. Transcriptome analysis confirmed the differences in the DNA repair pathways between the NS and MC cell lines. In addition, the NS-HL cell lines were radiosensitive and the MC-cell lines resistant to apoptosis after radiation exposure. Conclusions: In mononuclear NS-HL cells, loss of telomere integrity may present the first step in the ongoing process of chromosomal instability. Here, we identified, MSI as an additional mechanism for genomic instability in HL.

International audience; PURPOSE:To determine whether low dose/low dose rate radiation-induced genetic instability may result from radiation-induced inactivation of mechanisms induced by the ATM-dependent DNA damage response checkpoint. To this end, we analysed the faithfulness of T cell receptor (TR) gene rearrangement by V(D)J recombination in DNA from mice exposed to a single dose of X-ray or chronically exposed to low dose rate γ radiation.MATERIALS AND METHODS:Genomic DNA obtained from the blood or the thymus of wild type or Ogg1-deficient mice exposed to low (0.1) or intermediate/high (0.2-1 Gy) doses of radiation either by acute X-rays exposure or protracted exposure to low dose-rate γ-radiation was used to analyse by PCR the presence of illegitimate TR gene rearrangements.RESULTS:Radiation exposure does not increase the onset of TR gene trans-rearrangements in irradiated mice. In mice where it happens, trans-rearrangements remain sporadic events in developing T lymphocytes.CONCLUSION:We concluded that low dose/low dose rate ionizing radiation (IR) exposure does not lead to widespread inactivation of ATM-dependent mechanisms, and therefore that the mechanisms enforcing genetic stability are not impaired by IR in developing lymphocytes and lymphocyte progenitors, including BM-derived hematopoietic stem cells, in low dose/low dose rate exposed mice.

The increasing risk of acute large-scale radiological/nuclear exposures of population underlines the necessity of developing new, rapid and high throughput biodosimetric tools for estimation of received dose and initial triage. We aimed to compare the induction and persistence of different radiation exposure biomarkers in human peripheral blood in vivo. Blood samples of patients with indicated radiotherapy (RT) undergoing partial body irradiation (PBI) were obtained soon before the first treatment and then after 24 h, 48 h, and 5 weeks; i.e. after 1, 2, and 25 fractionated RT procedures. We collected circulating peripheral blood from ten patients with tumor of endometrium (1.8 Gy per fraction) and eight patients with tumor of head and neck (2.0-2.121 Gy per fraction). Incidence of dicentrics and micronuclei was monitored as well as determination of apoptosis and the transcription level of selected radiation-responsive genes. Since mitochondrial DNA (mtDNA) has been reported to be a potential indicator of radiation damage in vitro, we also assessed mtDNA content and deletions by novel multiplex quantitative PCR. Cytogenetic data confirmed linear dose-dependent increase in dicentrics (p < 0.01) and micronuclei (p < 0.001) in peripheral blood mononuclear cells after PBI. Significant up-regulations of five previously identified transcriptional biomarkers of radiation exposure (PHPT1, CCNG1, CDKN1A, GADD45, and SESN1) were also found (p < 0.01). No statistical change in mtDNA deletion levels was detected; however, our data indicate that the total mtDNA content decreased with increasing number of RT fractions. Interestingly, the number of micronuclei appears to correlate with late radiation toxicity (r2 = 0.9025) in endometrial patients suggesting the possibility of predicting the severity of RT-related toxicity by monitoring this parameter. Overall, these data represent, to our best knowledge, the first study providing a multiparametric comparison of radiation biomarkers in human blood in vivo, which have potential for improving biological dosimetry.

The research for high-throughput diagnostic tests for victims of radio/nuclear incidents remains ongoing. In this context, we have previously identified candidate genes that predict risk of late-occurring hematologic acute radiation syndrome (HARS) in a baboon model. The goal of the current study was to validate these genes after radiation exposure in humans. We also examined ex vivo relative to in vivo measurements in both species and describe dose-response relationships. Eighteen baboons were irradiated in vivo to simulate different patterns of partial- or total-body irradiation (TBI), corresponding to an equivalent dose of 2.5 or 5 Sv. Human in vivo blood samples were obtained from patients exposed to different dose ranges: diagnostic computerized tomography (CT; 0.004-0.018 Sv); radiotherapy for prostate cancer (0.25-0.3 Sv); and TBI of leukemia patients (2 × 1.5 or 2 × 2 Sv, five patients each). Peripheral whole blood of another five baboons and human samples from five healthy donors were cultivated ex vivo and irradiated with 0-4 Sv. RNA was isolated pairwise before and 24 h after irradiation and converted into cDNA. Gene expression of six promising candidate genes found previously by us in a baboon model ( WNT3, POU2AF1, CCR7, ARG2, CD177, WLS), as well as three genes commonly used in ex vivo whole blood experiments ( FDXR, PCNA, DDB2) was measured using qRT-PCR. We confirmed the six baboon candidate genes in leukemia patients. However, expression for the candidate gene FDXR showed an inverse relationship, as it was downregulated in baboons and upregulated in human samples. Comparisons among the in vivo and ex vivo experiments revealed the same pattern in both species and indicated peripheral blood cells to represent the radiation-responsive targets causing WNT3 and POU2AF1 gene expression changes. CCR7, ARG2, CD177 and WLS appeared to be altered due to radiation-responsive targets other than the whole blood cells. Linear dose-response relationships of FDXR, WNT3 and POU2AF1 using human ex vivo samples corresponded with human in vivo samples, suggesting that ex vivo models for in vivo dose estimates can be used over a wide dose range (0.001-5 Sv for POU2AF1). In summary, we validated six baboon candidate genes in humans, but the FDXR measurements underscored the importance of independent assessments even when candidates from animal models have striking gene sequence homology to humans. Since whole blood cells represented the same radiation-responsive targets for FDXR, WNT3 and POU2AF1 gene expression changes, ex vivo cell culture models can be utilized for in vivo dose estimates over a dose range covering up to 3.5 log scales. These findings might be a step forward in the development of a gene expression-based high-throughput diagnostic test for populations involved in large-scale radio/nuclear incidents.

Previous investigations in gene expression changes in blood after radiation exposure have highlighted its potential to provide biomarkers of exposure. Here, FDXR transcriptional changes in blood were investigated in humans undergoing a range of external radiation exposure procedures covering several orders of magnitude (cardiac fluoroscopy, diagnostic computed tomography (CT)) and treatments (total body and local radiotherapy). Moreover, a method was developed to assess the dose to the blood using physical exposure parameters. FDXR expression was significantly up-regulated 24 hr after radiotherapy in most patients and continuously during the fractionated treatment. Significance was reached even after diagnostic CT 2 hours post-exposure. We further showed that no significant differences in expression were found between ex vivo and in vivo samples from the same patients. Moreover, potential confounding factors such as gender, infection status and anti-oxidants only affect moderately FDXR transcription. Finally, we provided a first in vivo dose-response showing dose-dependency even for very low doses or partial body exposure showing good correlation between physically and biologically assessed doses. In conclusion, we report the remarkable responsiveness of FDXR to ionising radiation at the transcriptional level which, when measured in the right time window, provides accurate in vivo dose estimates.

Leukaemia is the prevailing neoplastic disorder of the hematopoietic system. Epidemiological analyses of the survivors of the Japanese atomic bombings show that exposure to ionising radiation (IR) can cause leukaemia. Although a clear association between radiation exposure and leukaemia development is acknowledged, the underlying mechanisms remain incompletely understood. A hemizygous deletion on mouse chromosome 2 (del2) is a common feature in several mouse strains susceptible to radiation-induced acute myeloid leukaemia (rAML). The deletion is an early event detectable 24h after exposure in bone marrow cells. Ultimately, 15-25% of exposed animals develop AML with 80-90% of cases carrying del2. Molecular mapping of leukaemic cell genomes identified a minimal deleted region (MDR) on chromosome 2 (chr2) in which a tumour suppressor gene, Sfpi1 is located, encoding the transcription factor PU.1, essential in haematopoiesis. The remaining copy of Sfpi1 has a point mutation in the coding sequence for the DNA-binding domain of the protein in 70% of rAML, which alters a single CpG sequence in the codon for arginine residue R235. In order to identify chr2 deletions and Sfpi.1/PU.1 loss, we performed array comparative genomic hybridization (aCGH) on a unique panel of 79rAMLs. Using a custom made CGH array specifically designed for mouse chr2, we analysed at unprecedentedly high resolution (1.4M array- 148bp resolution) the size of the MDR in low LET and high-LET induced rAMLs (32 X-ray- and 47 neutron-induced). Sequencing of Sfpi1/PU.1DNA binding domain identified the presence of R235 point mutations, showing no influence of radiation quality on R235 type or frequency. We identified for the first time rAML cases with complex del2 in a subset of neutron-induced AMLs. This study allowed us to re-define the MDR to a much smaller 5.5Mb region (still including Sfpi1/PU.1), identical regardless of radiation quality.

Studies of gene expression have proved important in defining the molecular mechanisms of radiation action and identifying biomarkers of ionizing radiation exposure and susceptibility. The full transcriptional response to radiation is very complex since it also involves epigenetic mechanisms triggered by radiation exposure such as modifications of expression of noncoding RNA such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) that have not been fully characterized. To improve our understanding of the transcriptional response to radiation, we simultaneously monitored the expression of ten protein-coding genes, as well as 19 miRNAs and 3 lncRNAs in a time- and dose-dependent manner in stimulated human T lymphocytes obtained from two healthy donors (C1 and C2) and one patient with ataxia telangiectasia (AT), which is a well characterized radiosensitivity disorder. After 2 Gy X irradiation, expression levels were monitored at time points ranging from 15 min up to 24 h postirradiation. The majority of genes investigated responded rapidly to radiation exposure, with the peak up-regulation (CDKN1A, SESN1, ATF3, MDM2, PUMA and GADD45A) or down-regulation (CCNB1) occurring 2-3 h postirradiation, while DDB2, FDXR and CCNG1 responded with slower kinetics reaching a peak of expression between 5 and 24 h. A significant modification of expression after radiation exposure was observed for miR-34a-5p and miR-182-5p, with an up-regulation occurring at late time points reaching two to threefold at 24 h. Differences between two donors in miR-182-5p response to radiation were detected: for C2, up-regulation reached a plateau-phase around 5 Gy, while for C1, up-regulation was at its maximum around 3 Gy and then decreased at higher doses. Among the three lncRNAs studied, TP53TG1 demonstrated a weak up-regulation, reaching a maximum of 1.5-fold at 24 h after radiation exposure. Conversely, FAS-AS1 was up-regulated up to fivefold by 5 Gy irradiation. Our results indicate that expression of the majority of protein-coding genes allows discrimination of the AT from healthy donors when analyzed at 2 h. However, differences in expression between AT and healthy donors are no longer detectable 24 h postirradiation although, interestingly, linear dose responses for some of the genes studied are obtained at this time point. Furthermore, our study shows that miRNAs miR-34a-5p and miR-182-5p are responsive to radiation exposure in a dose- and time-dependent manner. Additionally, to the best of our knowledge, this is the first study to report that FAS-AS1 lncRNA is up-regulated by radiation exposure in an ATM-dependent fashion in human T lymphocytes.

Background: The occurrence of widespread RNA and DNA sequence differences in the human transcriptome was reported in 2011. Similar findings were described in a second independent publication on personal omics profiling investigating the occurrence of dynamic molecular and related medical phenotypes. The suggestion that the RNA sequence variation was likely to affect disease susceptibility prompted us to investigate with a range of algorithms the amino acid variants reported to be present in the identified peptides to determine if they might be disease-causing. Results: The predictive qualities of the different algorithms were first evalu ated by using nonsynonymous single-base nucleotide polymorphism (nsSNP) datasets, using independently established data on amino acid variants in several proteins as well as data obtained by mutational mapping and modelling of binding sites in the human serotonin transporter protein (hSERT). Validation of the used predictive algorithms was at a 75% level. Using the same algorithms, we found that widespread RNA and DNA sequence differences were predicted to impair the func tion of the peptides in over 57% of cases. Conclusions: Our findings suggest that a proportion of edited RNAs which serve as templates for protein synthesis is likely to modify protein function, possibly as an adaptive survival mechanism in response to environmental modifications.

The purpose of this study was to quantify the poorly understood radiation doses to murine bone marrow and blood from whole-body fluorine 18 ((18)F)-fluorodeoxyglucose (FDG) positron emission tomography (PET), by using specific biomarkers and comparing with whole body external low dose exposures. Groups of 3-5 mice were randomly assigned to 10 groups, each receiving either a different activity of (18)F-FDG: 0-37MBq or whole body irradiated with corresponding doses of 0-300mGy X-rays. Blood samples were collected at 24h and at 43h for reticulocyte micronucleus assays and QPCR analysis of gene expression in peripheral blood leukocytes. Blood and bone marrow dose estimates were calculated from injected activities of (18)F-FDG and were based on a recommended ICRP model. Doses to the bone marrow corresponding to 33.43mGy and above for internal (18)F-FDG exposure and to 25mGy and above for external X-ray exposure, showed significant increases in radiation-induced MN-RET formation relative to controls (P

Salivary biomarkers have important potential to facilitate breakthroughs in epidemiologic studies, management of emergency situations, and detection and surveillance of diseases by medical staff. During the last decade, an increasing number of studies on salivary biomarkers have been published as a consequence of the impressive development of new high-throughput technologies. Here, we present a review of salivary biomarkers potentially useful in ionizing radiation (IR) research, particularly in molecular epidemiologic studies. Although several salivary biomarkers of cancer and other IR-associated diseases have been identified, few salivary biomarkers of exposure and no biomarker of susceptibility or effects specific to IR have been reported so far. Further studies are therefore needed to fully assess the potential of saliva as a source of biomarkers in the radiation research field. Although the use of saliva samples is not without drawbacks, it could represent an ideal noninvasive alternative to blood, particularly in children and in the context of large molecular epidemiology studies on the effects of low doses of IR, where, given the expected limited magnitude of effects, an extensive number of samples is required to reach statistical significance. See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.” Cancer Epidemiol Biomarkers Prev; 23(12); 2673–80. ©2014 AACR.

Ionizing radiation (IR) exposure of cells in vitro and in vivo triggers a complex cellular response amongst which modifications of gene expression have been consistently reported. Nevertheless, little is currently known about the transcriptionally responsive genes which play a role in the inflammation response. In order to improve our understanding of such transcriptional response to radiation in vivo, we simultaneously monitored the expression of 249 genes associated with the inflammation response over the course of the radiotherapy treatment in blood of patients treated for endometrial or head and neck cancer. We have identified genes whose transcriptional expression is either up-regulated (ARG1, BCL2L1) or down-regulated (MYC) several fold in vivo. These modifications were consistently detected across patients and further confirmed by Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR); they were specifically significant towards the end of the radiotherapy treatment, five weeks following the first radiation fraction and more pronounced in endometrial patients (respectively 2.9, 4.1 and 1.8 times). Importantly, in an attempt to correlate expression levels with normal tissue reaction to IR, we also identified three other genes CD40, OAS2 and CXCR1 whose expression level fluctuations during radiotherapy were more pronounced in patients developing late normal tissue responses to curative radiotherapy after the end of the radiotherapy treatment. Overall, we identified inflammation-associated genes which are promising biomarkers of ionizing radiation exposure and susceptibility to radiation-induced toxicity.

International audience; Purpose: RENEB, ‘Realising the European Network of Biodosimetry and Physical Retrospective Dosimetry,’ is a network for research and emergency response mutual assistance in biodosimetry within the EU. Within this extremely active network, a number of new dosimetry methods have recently been proposed or developed. There is a requirement to test and/or validate these candidate techniques and inter-comparison exercises are a well-established method for such validation. Materials and methods: The authors present details of inter-comparisons of four such new methods: dicentric chromosome analysis including telomere and centromere staining; the gene expression assay carried out in whole blood; Raman spectroscopy on blood lymphocytes, and detection of radiation-induced thermoluminescent signals in glass screens taken from mobile phones. Results: In general the results show good agreement between the laboratories and methods within the expected levels of uncertainty, and thus demonstrate that there is a lot of potential for each of the candidate techniques. Conclusions: Further work is required before the new methods can be included within the suite of reliable dosimetry methods for use by RENEB partners and others in routine and emergency response scenarios. © 2016 Crown Copyright. Published by Informa UK Limited, trading as Taylor & Francis Group.

The application of random effect models to different radiation biomarkers, including cytogenetic, protein-based, and gene-expression based biomarkers, is discussed. Explicit case studies are provided for the latter two scenarios, in which random effect models appear especially attractive as they can cope well with the large inter-individual variation which is typical for these biomarkers.

Recent epidemiology studies highlighted the detrimental health effects of exposure to low dose and low dose rate ionizing radiation (IR): nuclear industry workers studies have shown increased leukaemia and solid tumour risks following cumulative doses of

International audience; While the biological effects of high-dose-ionizing radiation on human health are well characterized, the consequences of low-dose radiation exposure remain poorly defined, even though they are of major importance for radiological protection. Lymphocytes are very radiosensitive, and radiation-induced health effects may result from immune cell loss and/or immune system impairment. To decipher the mechanisms of effects of low doses, we analyzed the modulation of the T-cell receptor gene repertoire in mice exposed to a single low (0.1 Gy) or high (1 Gy) dose of radiation. High-throughput T-cell receptor gene profiling was used to visualize T-lymphocyte dynamics over time in control and irradiated mice. Radiation exposure induces "aging-like" effects on the T-cell receptor gene repertoire, detectable as early as 1 month post-exposure and for at least 6 months. Surprisingly, these effects are more pronounced in animals exposed to 0.1 Gy than to 1 Gy, where partial correction occurs over time. Importantly, we found that low-dose radiation effects are partially due to the hematopoietic stem cell impairment. Collectively, our findings show that acute low-dose radiation exposure specifically results in long-term alterations of the T-lymphocyte repertoire.

International audience; Despite substantial experimental and epidemiological research, there is limited knowledge of the uranium-induce health effects after chronic low-dose exposures in humans. Biological markers can objectively characterize pathological processes or environmental responses to uranium and confounding agents. The integration of such biological markers into a molecular epidemiological study would be a useful approach to improve and refine estimations of uranium-induced health risks. To initiate such a study, Concerted Uranium Research in Europe (CURE) was established, and involves biologists, epidemiologists and dosimetrists. The aims of the biological work package of CURE were 1. To identify biomarkers and biological specimens relevant to uranium exposure; 2. To define standard operating procedures (SOPs); and 3. To set up a common protocol (logistic, questionnaire, ethical aspects) to perform a large-scale molecular epidemiologic study in uranium-exposed cohorts. An intensive literature review was performed and led to the identification of biomarkers related to 1. retention organs (lungs, kidneys and bone); 2. other systems/organs with suspected effects (cardiovascular system, central nervous system and lympho-hematopoietic system); 3. target molecules (DNA damage, genomic instability); and 4. high-throughput methods for the identification of new biomarkers. To obtain high-quality biological materials, SOPs were established for the sampling and storage of different biospecimens. A questionnaire was developed to assess potential confounding factors. The proposed strategy can be adapted to other internal exposures and should improve the characterization of the biological and health effects that are relevant for risk assessment. © 2017 by Radiation Research Society.

Exposure to ionising radiation can lead to an increased risk of cancer, particularly leukaemia. In radiation-induced acute myeloid leukaemia (rAML), a partial hemizygous deletion of mouse chromosome 2 is a common feature in several susceptible strains. The deletion is an early event detectable 24h after exposure in bone marrow cells using cytogenetic techniques. Expanding clones of bone marrow cells with chromosome 2 deletions can be detected less than a year after exposure to ionising radiation in around half of the irradiated mice. Ultimately, 15-25% of exposed animals develop AML. It is generally assumed that leukaemia originates in an early progenitor cell or haematopoietic stem cell, but it is unknown whether the original chromosome damage occurs at a similar frequency in committed progenitors and stem cells. In this study, we monitored the frequency of chromosome 2 deletions in immature bone marrow cells (Lin(-)) and haematopoietic stem cells/multipotent progenitor cells (LSK) by several techniques, fluorescent in situ hybridisation (FISH) and through use of a reporter gene model, flow cytometry and colony forming units in spleen (CFU-S) following ex vivo or in vivo exposure. We showed that partial chromosome 2 deletions are present in the LSK subpopulation, but cannot be detected in Lin(-) cells and CFU-S12 cells. Furthermore, we transplanted irradiated Lin(-) or LSK cells into host animals to determine whether specific irradiated cell populations acquire an increased proliferative advantage compared to unirradiated cells. Interestingly, the irradiated LSK subpopulation containing cells carrying chromosome 2 deletions does not appear to repopulate as well as the unirradiated population, suggesting that the chromosomal deletion does not provide an advantage for growth and in vivo repopulation, at least at early stages following occurrence.

To seek alterations in gene transcription in bone marrow cells following in vivo exposure of juvenile mice to power frequency magnetic fields, young (21–24-day old) C57BL/6 mice were exposed to a 100 μT 50 Hz magnetic field for 2 h. Transcription was analysed by three methods, High Coverage Expression Profiling (HiCEP), Illumina microarrays and quantitative real-time polymerase chain reaction (QRT-PCR). A pilot HiCEP experiment with 6 exposed (E) and 6 non-exposed (NE) mice identified four candidate responsive transcripts (two unknown transcripts (AK152075 and F10-NED), phosphatidylinositol binding clathrin assembly protein (Picalm) and exportin 7 (Xpo7)). A larger experiment compared 19 E and 15 NE mice using two independent QRT-PCR assays and repeated microarray assays. No significant field-dependent changes were seen, although Picalm showed a trend to significance in one QRT-PCR assay (E/NE = 0.91; P = 0.06). However, the study was underpowered to detect an effect of this magnitude (52% power at P = 0.05). These data indicate the current experimental constraints in detecting small changes in transcription that may occur in response to magnetic fields. These constraints result from technical limitations in the accuracy of assays and biological variation, which together were sufficient to account statistically for the number of differentially expressed transcripts identified in the pilot experiment.

The CBA/H mouse model of radiation-induced acute myeloid leukaemia (rAML) has been studied for decades to bring to light the molecular mechanisms associated with multistage carcinogenesis. A specific interstitial deletion of chromosome 2 found in a high proportion of rAML is recognised as the initiating event. The deletion leads to the loss of Sfpi, a gene essential for haematopoietic development. Its product, the transcription factor PU.1 acts as a tumour suppressor in this model. Although the deletion can be detected early following ionising radiation exposure by cytogenetic techniques, precise characterisation of the haematopoietic cells carrying the deletion and the study of their fate in vivo cannot be achieved. Here, using a genetically engineered C57BL/6 mouse model expressing the GFP fluorescent molecule under the control of the Sfpi1 promoter, which we have bred onto the rAML-susceptible CBA/H strain, we demonstrate that GFP expression did not interfere with X-ray induced leukaemia incidence and that GFP fluorescence in live leukaemic cells is a surrogate marker of radiation-induced chromosome 2 deletions with or without point mutations on the remaining allele of the Sfpi1 gene. This study presents the first experimental evidence for the detection of this leukaemia initiating event in live leukemic cells.

Exposure to ionizing radiation increases the incidence of acute myeloid leukemia (AML), which has been diagnosed in Japanese atomic bombing survivors, as well as patients treated with radiotherapy. The genetic basis for susceptibility to radiation-induced AML is not well characterized. We previously identified a candidate murine gene for susceptibility to radiation-induced AML (rAML): C-terminal binding protein (CTBP)-interacting protein (CTIP)/retinoblastoma binding protein 8 (RBBP8). This gene is essential for embryonic development, double-strand break (DSB) resection in homologous recombination (HR) and tumor suppression. In the 129S2/SvHsd mouse strain, a nonsynonymous single nucleotide polymorphism (nsSNP) in Ctip, Q418P, has been identified. We investigated the role of Q418P in radiation-induced carcinogenesis and its effect on CTIP function in HR. After whole-body exposure to 3 Gy of X rays, 11 out of 113 (9.7%) 129S2/SvHsd mice developed rAML. Furthermore, 129S2/SvHsd mouse embryonic fibroblasts (MEFs) showed lower levels of recruitment of HR factors, Rad51 and replication protein A (RPA) to radiation-induced foci, compared to CBA/H and C57BL/6 MEFs, isolated from rAML-sensitive and resistant strains, respectively. Mitomycin C and alpha particles induced lower levels of sister chromatid exchanges in 129S2/SvHsd cells compared to CBA/H and C57BL/6. Our data demonstrate that Q418P nsSNP influences the efficiency of CTIP function in HR repair of DNA DSBs in vitro and in vivo, and appears to affect susceptibility to rAML.

Scid hematopoietic stem cells (HSCs) have an intrinsic defect in their maintenance within the bone marrow (BM) niche which facilitates HSC transplantation without the absolute requirement of prior conditioning. Nevertheless, NOD scid mice have a significantly altered life span due to early development of thymic lymphomas, which compromises the ability to study the long-term fate of exogenous HSCs and their progeny. Here, we present data on the transplantation of HSCs into NOD scid gamma (NSG) mice to achieve long-term engraftment without prior conditioning. We transplanted allogeneic HSCs constitutively expressing the mCherry fluorescent marker into age-matched NSG mice and assessed donor chimerism 6 months post-transplantation. All transplanted NSG mice showed long-term myeloid and lymphoid cell chimerism. Also, in vivo irradiated HSCs showed long-term engraftment, although overall white blood cell (WBC) donor chimerism was lower compared with non-irradiated HSCs. Using this novel NSG transplantation model, we will be able to study the effects of low dose in vivo X-ray exposure on the long-term fate of HSCs, without the requirement of prior radio-ablation of the recipient, and thus leaving the recipient’s BM microenvironment uncompromised. In conclusion, we demonstrated for the first time that allogeneic HSCs from a different inbred strain can compete for niches in the BM compartment of NSG mice.

Ionizing radiation is a potent carcinogen, inducing cancer through DNA damage. The signatures of mutations arising in human tissues following in vivo exposure to ionizing radiation have not been documented. Here, we searched for signatures of ionizing radiation in 12 radiation-associated second malignancies of different tumour types. Two signatures of somatic mutation characterize ionizing radiation exposure irrespective of tumour type. Compared with 319 radiation-naive tumours, radiation-associated tumours carry a median extra 201 deletions genome-wide, sized 1–100 base pairs often with microhomology at the junction. Unlike deletions of radiation-naive tumours, these show no variation in density across the genome or correlation with sequence context, replication timing or chromatin structure. Furthermore, we observe a significant increase in balanced inversions in radiation-associated tumours. Both small deletions and inversions generate driver mutations. Thus, ionizing radiation generates distinctive mutational signatures that explain its carcinogenic potential., Ionizing radiation may induce irreparable DNA damage leading to cancer. Here, the authors identify a specific signature of mutations arising in patients exposed to ionizing radiation and suggest that radiation-induced tumorigenesis is associated with higher rates of genome-wide deletions and balanced inversions.

The potential health impacts of chronic exposures to uranium, as they occur in occupational settings, are not well characterized. Most epidemiological studies have been limited by small sample sizes, and a lack of harmonization of methods used to quantify radiation doses resulting from uranium exposure. Experimental studies have shown that uranium has biological effects, but their implications for human health are not clear. New studies that would combine the strengths of large, well-designed epidemiological datasets with those of state-of-the-art biological methods would help improve the characterization of the biological and health effects of occupational uranium exposure. The aim of the European Commission concerted action CURE (Concerted Uranium Research in Europe) was to develop protocols for such a future collaborative research project, in which dosimetry, epidemiology and biology would be integrated to better characterize the effects of occupational uranium exposure. These protocols were developed from existing European cohorts of workers exposed to uranium together with expertise in epidemiology, biology and dosimetry of CURE partner institutions. The preparatory work of CURE should allow a large scale collaborative project to be launched, in order to better characterize the effects of uranium exposure and more generally of alpha particles and low doses of ionizing radiation. © 2016 IOP Publishing Ltd.

// Christophe Badie 1 , Agnieszka Blachowicz 2 , Zarko Barjaktarovic 3 , Rosemary Finnon 1 , Arlette Michaux 4 , Hakan Sarioglu 5 , Natalie Brown 1 , Grainne Manning 1 , M. Abderrafi Benotmane 4 , Soile Tapio 3 , Joanna Polanska 2 , Simon D. Bouffler 1 1 Radiation Effects Department, Centre for Radiation, Chemical and Environmental Hazards, Public Health England, Chilton, UK 2 Faculty of Automatic Control, Electronics and Computer Science, Silesian University of Techology, Gliwice, Poland 3 Helmholtz Zentrum Munchen, German Research Center for Environmental Health GmbH, Radiation Proteomics Group, Institute of Radiation Biology, Neuherberg, Germany 4 Radiobiology Unit, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•.CEN), Mol, Belgium 5 Helmholtz Zentrum Munchen, German Research Center for Environmental Health GmbH, Research Unit Protein Science, Neuherberg, Germany Correspondence to: Simon D. Bouffler, email: simon.bouffler@phe.gov.uk Keywords: ionising radiation, acute myeloid leukaemia, mouse, gene expression, protein expression Received: February 11, 2016 Accepted: May 09, 2016 Published: May 26, 2016 ABSTRACT A combined transcriptome and proteome analysis of mouse radiation-induced AMLs using two primary AMLs, cell lines from these primaries, another cell line and its in vivo passage is reported. Compared to haematopoietic progenitor and stem cells (HPSC), over 5000 transcriptome alterations were identified, 2600 present in all materials. 55 and 3 alterations were detected in the proteomes of the cell lines and primary/ in vivo passage material respectively, with one common to all materials. In cell lines, approximately 50% of the transcriptome changes are related to adaptation to cell culture, and in the proteome this proportion was higher. An AML ‘signature’ of 17 genes/proteins commonly deregulated in primary AMLs and cell lines compared to HPSCs was identified and validated using human AML transcriptome data. This also distinguishes primary AMLs from cell lines and includes proteins such as Coronin 1, pontin/RUVBL1 and Myeloperoxidase commonly implicated in human AML. C-Myc was identified as having a key role in radiation leukaemogenesis. These data identify novel candidates relevant to mouse radiation AML pathogenesis, and confirm that pathways of leukaemogenesis in the mouse and human share substantial commonality.

Dysregulation of apoptosis is a key attribute of cancer, especially the one induced by p53 expression disruption. Radiotherapy, sometimes supported by chemotherapy and/or pre-surgery, is recommended in majority of cases, but despite of the very well defined treatment protocols and high quality irradiation procedure, the huge dispersion in response to the radiotherapy is observed among cancer patients. Patient radiosensitivity, according to up-to-date knowledge, is at least partially responsible for different reactions to ionising radiation. Here we concentrate on investigation of single nucleotide polymorphisms (SNP) which can possibly explain the radiation response phenomena. To reach this goal dependent and independent methods of p-value integrations are presented and compared. Both statistical and molecular function domains are used in comparison study. We propose a novel method of p-value integration which includes the control of gene expression trend and introduces the adaptive significance level. What is more the multigene approach is proposed in contrary to classical single gene investigation. As a result, set of statistically significant polymorphisms was obtained, among which some were identified as possible deleterious for KRAS signalling pathway.

The response to any radiation accident or incident involving actual or potential ionising radiation exposure requires accurate and rapid assessment of the doses received by individuals. The techniques available today for biodosimetry purposes are not fully adapted to rapid high-throughput measurements of exposures in large numbers of individuals. A recently emerging technique is based on gene expression analysis, as there are a number of genes which are radiation responsive in a dose-dependent manner. The present work aimed to assess a new technique which allows the detection of the level of expression of up to 800 genes without need of enzymatic reactions. In order to do so, human peripheral blood was exposed ex vivo to a range of x-ray doses from 5 mGy to 4 Gy of x-rays and the transcriptional expression of five radiation-responsive genes PHPT 1, PUMA , CCNG 1, DDB 2 and MDM 2 was studied by both the nCounter Digital Analyzer and Multiplex Quantitative Real-Time Polymerase Chain Reaction (MQRT-PCR) as the benchmark technology. Results from both techniques showed good correlation for all genes with R 2 values ranging between 0.8160 and 0.9754. The reproducibility of the nCounter Digital Analyzer was also assessed in independent biological replicates and proved to be good. Although the slopes of the correlation of results obtained by the techniques suggest that MQRT-PCR is more sensitive than the nCounter Digital Analyzer, the nCounter Digital Analyzer provides sensitive and reliable data on modifications in gene expression in human blood exposed to radiation without enzymatic amplification of RNA prior to analysis.

Radiation-induced acute myeloid leukaemias (AMLs) in mice are characterised by deletions and point mutations in the Sfpi1/PU.1 transcription factor. Six AML cell lines were used to examine the impact of three previously described R235 point mutations. AML cells carry myeloid and stem cell markers and the R235 mutations differentially affect mRNA and protein abundance. Expression of Sfpi1/PU.1 target genes was deregulated in a broadly similar fashion irrespective of R235 mutation including Flt3, which is frequently subject to activating mutations in human myeloid leukaemias. While R235 mutations differentially affect protein abundance they resulted in similar disruption of Sfpi1/PU.1 functions.

Genetic factors are likely to affect individual cancer risk, but few quantitative estimates of heritability are available. Public health radiation protection policies do not in general take this potentially important source of variation in risk into account. Two surrogate cellular assays that relate to cancer susceptibility have been developed to gain an insight into the role of genetics in determining individual variation in radiosensitivity. These flow cytometric assays for apoptosis induction and cell cycle delay following radiation are sufficiently sensitive to distinguish lymphocytes from a healthy donor population from those of a sample of obligate carriers of ATM mutations (P = 0.01 and P = 0.02, respectively). Analysis of 54 unselected twin pairs (38 dizygotic, 16 monozygotic) indicated much greater intrapair correlation in response in monozygotic than in dizygotic pairs. Structural equation modelling indicated that models including unique environmental factors only fitted the data less well than those incorporating two or more of additive genetic factors, common environmental factors and unique environmental factors. A model incorporating additive genetic factors and unique environmental factors yielded estimates of heritability for the two traits of 68% (95% CI 40-82%, cell cycle) and 59% (95% CI 22-79%, apoptosis). Thus, these data suggest that genetic factors contribute significantly to human variation in these two measures of radiosensitivity that relate to cancer susceptibility.

The Ku heterodimer is a DNA end-binding protein that promotes the non-homologous end joining (NHEJ) pathway of DNA double strand break (DSB) repair by recruiting the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). Ku is also a normal component of telomeres where it is required for telomere maintenance, interacting not only with the DNA but also with various telomere proteins including telomerase. The way in which Ku simultaneously plays such distinct roles, end-joining at DSBs and end-maintenance at telomeres, is unclear. One way to address this is to study cells in which the NHEJ and telomeric roles of Ku have been separated. Here we describe human cells that express fusions between the large human Ku subunit (Ku86) and a fluorescent protein tag. These cells have reduced telomerase activity and increased sensitivity to ionizing radiation (IR) but no change in their DNA-PK activity or in the DNA end-binding of endogenous Ku. Cells with particularly large amounts of one fusion protein undergo progressive telomere shortening and cellular senescence. These data are consistent with models in which Ku recruits telomerase to telomeres or activates recruited telomerase and suggest that the Ku86 fusion proteins specifically block this role.

The aim of this study is to verify the effectiveness of a statistical integrative approach for merging expression data sets on the level of p-values. The data consist of two independent sets of expression levels for breast cancer patient lymphocyte tissue. The samples represent two groups: sensitive and resistant to the impact of ionizing radiation. Three approaches for integrating information derived from the two experiments to select a radiosensitivity gene signature were investigated: restrictive, non-statistical and integrative. Signature validity was assessed by verifying data separability using the support vector machine procedure and a logistic regression model selected using the likelihood ratio test to account for regularization. We demonstrated the value of additional information retained in the statistical method of gene selection based on combined p-values, which as the only logistic regression model in its optimal setting attained 100 % separability (AUC 86.2 % restrictive and 85.6 % non-statistical). Moreover, for the best support vector machine classifier, our p-value combination approach outperformed the restrictive and Arraymining methods (AUC 96.7 % versus 87.9 % and 94.6 % respectively). Further functional validation by signaling pathway research proved conjointly the biological relevance of the supreme gene signature.

The mapping of genes which affect individual cancer risk is an important but complex challenge. A surrogate assay of susceptibility to radiation-induced acute myeloid leukaemia (AML) in the mouse based on chromosomal radiosensitivity has been developed and validated. This assay was applied to the mapping of radiation-induced AML risk modifier loci by association with microsatellite markers. A region on chromosome (chr) 18 with strong association is identified and confirmed by backcross analysis. Additional loci on chrs 8 and 13 show significant association. A key candidate gene Rbbp8 on chr18 is identified. Rbbp8 is shown to be upregulated in response to X-irradiation in the AML sensitive CBA strain but not AML resistant C57BL/6 strain. This study demonstrates the strength of utilizing surrogate endpoints of cancer susceptibility in the mapping of mouse loci and identifies additional loci that may affect radiation cancer risk.

The transcription factor PU.1, encoded by the murine Sfpi1 gene (SPI1 in humans), is a member of the Ets transcription factor family and plays a vital role in commitment and maturation of the myeloid and lymphoid lineages. Murine studies directly link primary acute myeloid leukaemia (AML) and decreased PU.1 expression in specifically modified strains. Similarly, a radiation-induced chromosome 2 deletion and subsequent Sfpi1 point mutation in the remaining allele lead to murine radiation-induced AML. Consistent with murine data, heterozygous deletion of the SPI1 locus and mutation of the -14kb SPI1 upstream regulatory element were described previously in human primary AML, although they are rare events. Other mechanisms linked to PU.1 downregulation in human AML include TP53 deletion, FLT3-ITD mutation and the recurrent AML1-ETO [t(8;21)] and PML-RARA [t(15;17)] translocations. This review provides an up-to-date overview on our current understanding of the involvement of PU.1 in the initiation and development of radiation-induced AML, together with recommendations for future murine and human studies.

Background The identification of polymorphisms and/or genes responsible for an organism's radiosensitivity increases the knowledge about the cell cycle and the mechanism of the phenomena themselves, possibly providing the researchers with a better understanding of the process of carcinogenesis. Aim The aim of the study was to develop a data analysis strategy capable of discovering the genetic background of radiosensitivity in the case of small sample size studies. Results Among many indirect measures of radiosensitivity known, the level of radiation-induced chromosomal aberrations was used in the study. Mathematical modelling allowed the transformation of the yield-time curve of radiation-induced chromosomal aberrations into the exponential curve with limited number of parameters, while Gaussian mixture models applied to the distributions of these parameters provided the criteria for mouse strain classification. A detailed comparative analysis of genotypes between the obtained subpopulations of mice followed by functional validation provided a set of candidate polymorphisms that might be related to radiosensitivity. Among 1857 candidate relevant SNPs, that cluster in 28 genes, eight SNPs were detected nonsynonymous (nsSNP) on protein function. Two of them, rs48840878 (gene Msh3) and rs5144199 (gene Cc2d2a), were predicted as having increased probability of a deleterious effect. Additionally, rs48840878 is capable of disordering phosphorylation with 14 PKs. In silico analysis of candidate relevant SNP similarity score distribution among 60 CGD mouse strains allowed for the identification of SEA/GnJ and ZALENDE/EiJ mouse strains (95.26% and 86.53% genetic consistency respectively) as the most similar to radiosensitive subpopulation Conclusions A complete step-by-step strategy for seeking the genetic signature of radiosensitivity in the case of small sample size studies conducted on mouse models was proposed. It is shown that the strategy, which is a combination of mathematical modelling, statistical analysis and data mining methodology, allows for the discovery of candidate polymorphisms which might be responsible for radiosensitivity phenomena.

The aim of the study was to develop a data analysis strategy capable of discovering the genetic background of radiosensitivity. Radiosensitivity is the relative susceptibility of cells, tissues, organs or organisms to the harmful effect of radiation. Effects of radiation include the mutation of DNA specialy in genes responsible for DNA repair. Identification of polymorphisms and genes responsible for an organisms’ radiosensitivity increases the knowledge about the cell cycle and the mechanism of radiosensitivity, possibly providing the researchers with a better understanding of the process of carcinogenesis. To obtain this results, mathematical modeling and data mining methods were used.