Issue 1

Newsletter
Studies on a cohort of Serbian children exposed to x-irradiation to determine the
contribution of the non-coding genome to susceptibility at low doses
The Dark.Risk project is
financially supported by
the 7th Framework
programme of the
European Commission.

Welcome
The Dark.Risk project has now been running for one year, with a European researcher
team. We welcome you to this first issue of Dark.Risk newsletter. This will be bi-annual.
The newsletter is launched to reach a wider audience seeking information about the
project. We look forward to launching useful and informative issues. We welcome any
feedback and contributions.

Short introduction of the project

In the late 1950s public health initiatives in several European

countries mandated scalp depilation as the standard of care
for the treatment for children infected with a fungal disease
of the hair roots (Tinea capitis). The standard treatment
protocol used a mild dose of X-irradiation to the head to kill
hair cells, and proved highly effective in combating the
fungus. Only decades later it was noted, firstly in Israel, then
in the USA and other countries, that there was an increased frequency of cancer in the
treated individuals.

Inside this issue

Main Objective

p2

First reults
WP 1

ROSA

p3

WP2/3 SERGAS

p2

HMGU

p3

ENEA

p4

UAB

p4

Meeting & Events

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Website

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Partners

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The long term health effects following exposure to low and

moderate ionizing radiation remain uncertain. At doses
likely to be received from medical diagnostic procedures
(e.g. Computer Tomography (CT) or Positron Emission
Tomography (PET)) there is a lack of convincing evidence
for or against a risk to health. Within the Dark.Risk project
we have the unique opportunity to study long-term health
effects in a large cohort of individuals (~ 25.000) exposed
during childhood to a highly uniform dose of radiation to the head and to create the
Serbian Registry of Tinea capitis children (SRTCC).
It is the mission of Dark.Risk to evaluate the potential of the non-coding genome
to deliver information on disease outcome and association with radiation.

Facts and figures

Total cost____________2.3 M
EC contribution_______1.7M
Duration_________36 Months

From______2012-10-01
To________2015-09-30
Website____Dark.Risk

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Main objective
Dark.Risk will prepare the way to use SRTCC biomaterials to quantify
the contribution of individual differences in sensitivity to risk from
low doses of ionizing radiation. The focus will be on a novel and
unique process to identify individual differences. Until recently 95%
of the human genome was considered to be non-translated junk"
DNA. However, almost all of this dark matter is actively transcribed
into a variety of non-coding RNA species (ncRNAs) that regulate
interactions with the external environment, such as repressing
retrotransposons, regulating gene transcription responses to stress,
governing chromatic remodeling and coordinating the translation of mRNAs. The ncRNAs play an essential role
in coordinating critical cellular responses to radiation such as maintenance of genomic stability, senescence,
apoptosis and survival.
The goal of Dark.Risk is to establish a proof of concept by analysing the health status of Tinea capitis treated Serbian
children with the aim of obtaining information on long-term health effects of low dose radiation exposure.

First results
WP 1
Within the Dark.Risk project, Work Package 1 is
main responsibility of ZENSKA ASOCIJACIJA
"ROSA" team.
WP1 is designed to firstly establish the Serbian
Registry of Tinea Capitis Children. This will be
followed by the recruitment of the cohort for
epidemiological investigation on late health
consequences related to TC treatment, that is applied X-ray irradiation used in inducing scalp hair
removal. Biological samples will then be collected from a selected number of individuals in this cohort, and
analysed for targeted genetic traits by other Dark.Risk partners.
At the end of year one, ROSA's work can be briefly recapitulated as follows. In this period the primary
objective was to carry out identification of patients registered at the Belgrade Mycosis Hospital from 1950st
1959. It was planned that 8,000 individuals from the hospital records be identified by October 1 2013. This
number has been reached, and we expect that it will continue to rise steadily, as scheduled by dynamics of
data collection. All information concerning patients' identity is stored into a digital database and is ready
to be secured at the Serbian Institute of Health. In accord with Serbian legislation Act on Conducting
Medical Research these records will be held there on behalf of ROSA.
We believe that this year was truly successful to us, and we hope that further stages of our work will
continue to progress in an equally successful manner, meeting objectives and timetables of Dark.Risk.

WP 2/3
SERGAS
Previous studies of the members of this consortium showed that reduced expression of Rb1 prevents
radiation-induced cell cycle arrest and simultaneously creates shortening of the telomeres leading to an
increase in genomic instability (Gonzalez-Vasconcellos et al., Cancer Research, 2013).
These findings were linked to the changes in expression of a telomere specific long non coding RNA
(lncRNA) called TERRA. Together these two activities can explain the increase in susceptibility to
radiation-induced cancer in Rb1 insufficiency.

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During this first Dark.Risk year, the group of Dr. Fernndez

(part of WP2 and WP3) is studying how Rb1 regulates
transcription of the TERRA lncRNA, its action at the level of the
TERRA promoter and the possible telomere epigenetic alterations
arising from mutations in the Rb1 tumour suppressor gene.
They are setting up an assay (see figure) to investigate the
regulation of TERRA expression in cells lacking one copy of the
Rb1 gene. Chromosomes 15, 10 and X/Y (in human) and 18, 10 and
X/Y (in mouse) are being analysed at their subtelomeric regions
ensuring that the studies on TERRA transcriptional levels are both telomere length and locationindependent. Furthermore, a standardisation of Q-PCR methods to measure telomeric length in a large
number of samples is being performed.
Finally, a new technique to discriminate between different epigenetic states of telomeres assayed at the
chromatin condensation level is being developed and evaluated.

HMGU
Although, the data of the radiation effects on non-coding RNA transcripts (lncRNAs, miRNAs) and deeper
epigenetical control are in their infancy, we could confirmed the importance of miRNAs modulation in
cells after irradiation. HMGU reports here the expression profiling of the radiation response of ~15,000
lncRNAs that was undertaken with human microarrays (ArrayStar) using RNA from EA.hy926 (human
endothelial cell line), U2OS (human osteosarcoma tumour cell line) and T47D (human breast epithelial
tumour cell line), 4hr after 2.5Gy gamma irradiation exposure.
Differentially expressed lncRNAs were identified
following quantile normalisation, fold change filter
processing (GeneSpring) with sample and control
(sham irradiated) comparison. Of the seven regulated
lncRNAs that were subsequently selected for
validation, all exhibited the same up- or downregulation following irradiation in each of the three
cell lines. Consequently an lncRNA that shows strong
up-regulation in EA.hy926 cells was also found upregulated in T47D and U2OS cells, and vice versa.
This is the first indication that a given radiationregulated lncRNA has a defined role across different
cell lines during the radiation response. Such a result
is a strong contrast to the situation observed for
miRNAs, which exhibit highly cell-type specific
changes in expression.
Mean regulations of more than 2-fold up-regulation
were found for the lncRNAs PANDA and linc-p21. Significantly altered lncRNAs associated with genes
involved in cancer were selected for Taqman real-time RT-PCR verification in T47D and MDA-MB-361
breast cancer cell lines 4hr and 24hr after 0.25Gy and 2.5Gy irradiation. Although many long non-coding
RNAs (lncRNAs) are deregulated, the characterisation of the majority still remains unresolved. The
genomic context of lncRNAs can assist in predicting their functional role. One previously uncharacterized
lncRNA (abbreviated rad-lncRNA1) was highly expressed in T47D and MDA-MB-361 cells 24hr after low
dose (0.25Gy) irradiation exposure. These results suggest a potential role of lncRNAs in the regulation of
specific gene expression with implications for epigenetic control of low dose radiation effects.

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ENEA
Studies by ENEA resulted in following: Medulloblastoma (MB) is the most common malignant brain
tumour of children, arising from cerebellar granule precursors (CGPs). Large fraction of human MBs
reveals mutations in SHH pathway, one of the major regulators of cerebellar development. Patched1
knockout mice (Ptch1+/-), one of the most studied models of medulloblastoma due to SHH pathway
deregulation, represent an ideal in vivo model to investigate the role of ionizing radiation in MB
development. Recent data demonstrate the relevance of miroRNAs in setting DNA damage response.
Therefore the Ptch1+/-mice is a useful model for studies of changes in the transcription profile of noncoding RNAs after irradiation.
ENEA carried out experimental settings to investigate the response to damage induced by radiation at
postnatal age 2 (P2). CGPs, isolated from Ptch1+/- and wt mouse cerebella, were irradiated with 0.1, 0.5, 1
Gy. After 4 hours, cells were stained with propidium iodine (PI) and subjected to flow cytometry to
determine their DNA content. Results show a statistically significant decrease of hypodiploid DNA in
mutant irradiated CGPs compared to their wt counterparts, for all experimental conditions, suggesting
that a lower rate of apoptosis could be caused by a more efficient DNA repair after damage. These data
comply with the increased proliferation index (determined by % S phase plus % of G2/M phases) managed
by activation of SHH pathway in the mutant CGPs compared to wt.
ENEA collected mutant and wt CGPs at P2 after irradiation with 1Gy of X-rays and performed the miRNA
PCR Arrays. This arrays profile the expression of 84 miRNAs known to alter their expression during
nervous system-related carcinogenesis. Preliminary data show various microRNAs modulated in mutant
GCPs versus wt. Some of the identified microRNAs are found deregulated in human medulloblastomas,
supporting the hypothesis that could be involved in this pathogenesis.

UAB
During this first year, at UAB we established a list of 32
long intergenic non-coding RNAs (lincRNAs) induced in
Breast Primary Epithelial Cells (BPECs) after a low or
moderate dose of X-ray exposure. The biological material
was obtained from normal breast tissue from disease-free
reduction mammoplasties. Cells were irradiated using a
mammogram X-ray device and a Therapax, which is an Xray source that is used to irradiate at higher doses but
emulated the dose rate and the energy given by the
mammogram. After irradiation, RNA was extracted and
hybridized in SurePrint G3 Human Gene Expression v2
Microarray, which includes probes to lincRNAs catalogued
by scientists at the Broad Institute.
Moreover, we investigated whether the human
homologous region of the murine lincRNA-p21 was
activated after irradiation. The lincRNA-p21 is activated
after doxorubicin treatment in mouse. It acts as a repressor of genes normally repressed by p53 (Huarte et
al 2010). Different primers covering all the human lincRNA-p21 sequence known so far were designed to
check its expression by qPCR after exposure to 2Gy of gamma-rays. No significant results were obtained
and thus we can conclude that radiation-induced damage does not activate its expression in BPECs and in
human dermal fibroblasts.

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Meetings & events

Contact details:

Kick-Off Meeting 6th to 7th November 2012 at HMGU

dark.risk@helmholtzmuenchen.de

The kick-off meeting was the first opportunity after the preparation of the project
application for all the partners to meet in person and to acquaint themselves in detail with
all the project tasks and the timelines. The Leader of each Work Package presented its
activities.

First annual meeting

will be held in La Corua, Spain from 6

th

th

to 7 November in 2013.

Website
The website is an instrument that allows communication
between all partners and makes the project known to the
scientific community and interested public, respectively.
It meets the necessary need to transfer the results and
specific knowledge arising from the research to a large
audience.
http://www.helmholtz-muenchen.de/isb/darkrisk

Literature
Gonzalez-Vasconcellos I, Anastasov N, Sanli-Bonazzi B, Klymenko O, Atkinson MJ, Rosemann M: Rb1 haploinsufficiency promotes telomere attrition and radiation-induced genomic instability. Cancer Res 2013 Jul 15;73(14):
4247-55.
Hernndez L, Terradas M, Martn M, Tusell L, Genesc A. Highly Sensitive Automated Method for DNA Damage
Assessment: Gamma-H2AX Foci Counting and Cell Cycle Sorting. International Journal of Molecular Sciences.
2013; 14(8):15810-15826.

Partners
The Dark.Risk group consists of 5 participating institutions (HMGU, ROSA, ENEA, UAB and
SERGAS) based in 4 different EU member states.
HELMHOLTZ ZENTRUM MUENCHEN, DEUTSCHES
FORSCHUNGSZENTRUM FUER GESUNDHEIT UND UMWELT GMBH
http://www.helmholtz-muenchen.de/

ZENSKA ASOCIJACIJA "ROSA"

http://www.humanas.rs/index.php?lang=en&main=womens-association-rosa

AGENZIA NAZIONALE PER LE NUOVE TECNOLOGIE,

L'ENERGIA E LO SVILUPPO ECONOMICO SOSTENIBILE
http://utbiorad.casaccia.enea.it/

UNIVERSITAT AUTONOMA DE BARCELONA

http://www.uab.es/

SERVIZO GALEGO DE SAUDE

http://www.sergas.es/