Two quality controlled inter-laboratory exercises were organized within the EU project 'Realizing the European Network of Biodosimetry (RENEB)' to further optimize the dicentric chromosome assay (DCA) and to identify needs for training and harmonization activities within the RENEB network.The general study design included blood shipment, sample processing, analysis of chromosome aberrations and radiation dose assessment. After manual scoring of dicentric chromosomes in different cell numbers dose estimations and corresponding 95% confidence intervals were submitted by the participants.The shipment of blood samples to the partners in the European Community (EU) were performed successfully. Outside the EU unacceptable delays occurred. The results of the dose estimation demonstrate a very successful classification of the blood samples in medically relevant groups. In comparison to the 1st exercise the 2nd intercomparison showed an improvement in the accuracy of dose estimations especially for the high dose point.In case of a large-scale radiological incident, the pooling of ressources by networks can enhance the rapid classification of individuals in medically relevant treatment groups based on the DCA. The performance of the RENEB network as a whole has clearly benefited from harmonization processes and specific training activities for the network partners.
Reliable dose estimation is an important factor in appropriate dosimetric triage categorization of exposed individuals to support radiation emergency response.Following work done under the EU FP7 MULTIBIODOSE and RENEB projects, formal methods for defining uncertainties on biological dose estimates are compared using simulated and real data from recent exercises.The results demonstrate that a Bayesian method of uncertainty assessment is the most appropriate, even in the absence of detailed prior information. The relative accuracy and relevance of techniques for calculating uncertainty and combining assay results to produce single dose and uncertainty estimates is further discussed.Finally, it is demonstrated that whatever uncertainty estimation method is employed, ignoring the uncertainty on fast dose assessments can have an important impact on rapid biodosimetric categorization.
Vral, A., Thierens, H., Baeyens, A. and De Ridder, L. The Micronucleus and G2-Phase Assays for Human Blood Lymphocytes as Biomarkers of Individual Sensitivity to Ionizing Radiation: Limitations Imposed by Intraindividual Variability. Radiat. Res. 157, 472–477 (2002).As part of a program to assess the applicability of the micronucleus (MN) and G2-phase assays as biomarkers of cancer susceptibility, we investigated the inter- and intraindividual variations of these end points. For the MN assay, unstimulated blood cultures from 14 healthy donors were exposed in vitro to 3.5 Gy 60Co γ rays; for the G2-phase assay, PHA-stimulated cell cultures were irradiated with a dose of 0.4 Gy 60Co γ rays in the G2 phase of the cell cycle. Two of the 14 volunteers were assayed 9 times over a period of 1 year. The repeat experiments revealed that the intraindividual variability was not significantly different from the interindividual variability for both the G2-phase and MN assays. Since the intraindividual variability determines the reproducibility of the assay, our results highlight the limitations of these end points in detecting reproducible differences in radiation sensitivity between individuals within a normal population. For example, one donor of the population was identified as being radiosensitive (based on the 90th percentile criterion) but turned out to be normal when the assay was repeated twice. We conclude that the determination of individual radiosensitivity with these two cytogenetic assays is unreliable when based on one blood sample.
Introduction: Primary immunodeficiencies (PIDs) are a heterogeneous group of disorders caused by genetically determined defects of the
immune system, predisposing to life-threatening complications such as severe and recurrent infections, auto-immunity and malignancies. A
subset of PIDs is caused by pathogenic germline variants in DNA repair genes. As a consequence these patients are radiosensitive and
present with increased cancer risk. Since PID patients are exposed to radiation for several reasons (bone marrow transplantation,
radiotherapy, diagnostic imaging), identification of the genetic defect is important for risk stratification and improved therapeutic
management. As in only 5-20% of the patients pathogenic variants are found in currently known PID genes, we hypothesize that defects in
additional DNA damage response genes may be involved in PID patients prone to malignancies.Materials and Methods: WES data is
analyzed by a DNA damage response panel consisting of +/- 1230 genes. Extensive filtering results in a long list of variants of unknown
clinical significance (VUS). To facilitate variant prioritization we are complementing WES with transcriptomics on RNA extracted from short
term lymphocyte cultures. We search for expression and splicing outliers in the transcriptome.Results: Using this approach we identified a
homozygous intronic variant, outside the canonical splice sites, in a DNA repair gene not previously linked to PID. RNA-seq revealed out-offrame
exon skipping. Further functional validations to establish a link with the phenotype are ongoing.Conclusions: These first findings
encourage the implementation of transcriptomics in the workup of PID patients to improve diagnosis and patient management.
Introduction
Primary immunodeficiencies (PIDs) are a heterogeneous group of disorders caused by genetically determined defects of the immune system, predisposing to life-threatening complications such as severe and recurrent infections, auto-immunity and malignancies. A subset of PIDs is caused by pathogenic germline variants in DNA repair genes. As a consequence these patients are radiosensitive and present with increased cancer risk. Since PID patients are exposed to radiation for several reasons (bone marrow transplantation, radiotherapy, diagnostic imaging), identification of the genetic defect is important for risk stratification and improved therapeutic management. As in only 5-20% of the patients pathogenic variants are found in currently known PID genes, we hypothesize that defects in additional DNA damage response genes may be involved in PID patients prone to malignancies. We applied an integrated approach of whole exome sequencing (WES) and transcriptomics to identify the underlying genetic defect in a patient with primary dwarfism, facial dysmorphia, skeletal abnormalities, severe microcephaly, mental retardation and immunodeficiency.
Materials and Methods
WES data is analyzed by means of a PID gene panel (460 genes) and a DNA damage response panel consisting of >1230 genes. Extensive filtering results in a long list of variants of unknown clinical significance (VUS). To facilitate variant prioritization we are complementing WES with transcriptomics on RNA extracted from short term lymphocyte cultures. We search for expression and splicing outliers in the transcriptome.
Results
WES did not reveal a pathogenic variant in the PID gene panel, but a homozygous variant at position +5 in intron 5 of the DNA repair gene ATRIP was found in the patient. RNA-Seq showed skipping of exon 5, leading to a premature stop codon. This was confirmed using Sanger sequencing at the cDNA level. No full length ATRIP transcripts were detected. Additionally western blot analysis shows complete absence of ATRIP protein in our patient.
Our patient only is the second ATRIP-deficient patient. The first patient ((Ogi et al. 2012)) had a clinical diagnosis of Seckel syndrome. Clinically our patient resembles SS, but additionally presents with an immunodeficiency. Previously a link between this gene and PID has not been established. Further functional validations are ongoing to provide insights into the link with the immune phenotype.
Conclusions
In summary, these first findings encourage the complementation of a PID gene panel with a panel of DNA damage genes for the molecular work-up of PID patients with a (presumed) cancer predisposition. Furthermore, transcriptomics allows prioritization of variants and improves diagnosis and patient management. We expanded the molecular and clinical spectrum of Seckel syndrome by describing the second ATRIP-deficient patient. Further functional work-up will contribute to the disease mechanism.
Purpose: In many countries, breast cancer screening programs based on periodic mammography exist, giving a large group of women regularly a small dose of ionizing radiation. In order to assess the benefit/risk ratio of those programs the relative biological effectiveness (RBE) of mammography X-rays needs to be determined.Materials and methods: Blood of five healthy donors was irradiated in vitro with 30 kV X-rays and 60Co γ-rays with doses between 5 and 2000 mGy. The phosphorylated histone subtype H2A isoform X-foci (γH2AX-foci) technique was used to quantify the number of DNA double-strand breaks (DSB) after irradiation. Chromosomal damage resulting from non- or misrepaired DNA DSB was quantified with the micronucleus (MN)-assay and the sensitivity was improved by counting only centromere negative micronuclei (MNCM−).Results: The threshold detection dose obtained with the γH2AX-foci test was 10 mGy for mammography X-rays compared to 50 mGy for γ-rays. With the MN-assay respectively MN-centromere-assay threshold detection doses of 100, respectively, 50 mGy were obtained for mammography X-rays compared to 200 respectively 100 mGy for γ-rays. An RBE of 1.4 was obtained with the γH2AX-foci assay. With the MN-assays low-dose RBE values between 3 and 4 were determined.Conclusion: Our results indicate that exposure to mammography X-rays resulted in a modest increase in the induction of DSB compared to γ-rays. However, due to the higher linear energy transfer (LET) of mammography X-rays more clustered DNA damage is produced that is more difficult to repair and results in a more pronounced increase in micronucleus formation.
Purpose: Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G0-lymphocyte PCC for harmonization, standardization and optimization of the PCC assay.Materials and methods: Comparative analysis among different partners for dose assessment included shipment of PCC-slides and captured images to construct dose-response curves for up to 6 Gy γ-rays. Accident simulation exercises were performed to assess the suitability of the PCC assay by detecting speed of analysis and minimum number of cells required for categorization of potentially exposed individuals.Results: Calibration data based on Giemsa-stained fragments in excess of 46 PCC were obtained by different partners using galleries of PCC images for each dose-point. Mean values derived from all scores yielded a linear dose-response with approximately 4 excess-fragments/cell/Gy. To unify scoring criteria, exercises were carried out using coded PCC-slides and/or coded irradiated blood samples. Analysis of samples received 24 h post-exposure was successfully performed using Giemsa staining (1 excess-fragment/cell/Gy) or centromere/telomere FISH-staining for dicentrics.Conclusions: Dose assessments by RENEB partners using appropriate calibration curves were mostly in good agreement. The PCC assay is quick and reliable for whole- or partial-body triage biodosimetry by scoring excess-fragments or dicentrics in G0-lymphocytes. Particularly, analysis of Giemsa-stained excess PCC-fragments is simple, inexpensive and its automation could increase throughput and scoring objectivity of the PCC assay.
