Page 381 - 2014 Printable Abstract Book
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cell line. Two clones were selected from the 0 Gy control groups, 2 from 2 Gy exposed groups, and 3 from
4 Gy irradiated groups, and DNA recovered from each clone. We confirmed each clone as derived from
single cell by detecting rearrangement patterns of the JH region in the IGH gene. Whole genome
sequencing of DNA from the 7 clones and DNA from the original line was carried out at the Beijing
Genomics Institute by the 90 bp paired-end method using Hiseq2000. After obtaining 90 Gb of raw
sequence data per sample, we mapped them to human reference sequence GRCh37/hg19 by BWA and
analyzed using samtools and in-house programs to identify mutation candidates. We judged the change
of sequences as mutation candidates when observed only in one clone but not observed in other clones,
the original cell line, and the reference sequence. We identified 12 relatively large deletions in control and
irradiated clones consistent with those identified by microarray CGH analyses previously conducted in our
laboratory. Translocation and inversion type mutations were also identified only in the exposed group.
Approximately 20-30 small (<5 bp) indel mutation candidates were also found in control and irradiated
clones. The majority of candidates involved simple repeat sequences such as AAAA to AAA or GCGC to
GCGCGC. Among indels in unique sequences, only from 3-9 candidates per clone were able to be
confirmed as true mutations with Sanger sequencing. We also identified ~1,500 mutation candidates
involving single nucleotide substitutions per clone. Using a stringent filtering (variant frequency >0.3,
depth of coverage > 20, and locating at non-repetitive region for enabling a validation by Sanger
sequencing), the number of the true base substitution mutationswas estimated to be 100-400 per clone.
Based on these data we found that the number of indel- and base-substitution mutations was increased
following irradiation.
(PS7-50) Induction and repair of clustered DNA double strand breaks in human fibroblasts after
1
4
1;2
1;3
irradiation with Boron ions and γ-rays. Lucie Jezkova ; Alla Boreyko ; Tatiana Bulanova ; Martin Falk ;
1;3
1;3
4
4
5
Iva Falkova ; Marie Davidkova ; Stanislav Kozubek ; Evgeny Krasavin ; Elena Kruglyakova ; Olga
2
1;3
1
Valentova ; and Mariia Zadneprianetc , Joint Institute for Nuclear Research, Dubna, Russian Federation ;
2
Institute of Chemical Technology Prague, Prague, Czech Republic ; "Dubna" International University for
3
Nature, Society, and Man, Dubna, Russian Federation ; Institute of Biophysics, Academy of Sciences of the
4
Czech Republic, Brno, Czech Republic ; and Nuclear Physics Institute, Academy of Sciences of the Czech
Republic, Prague, Czech Republic
5
The nature of the damage in DNA caused by the accelerated charged particles significantly differs
from the damage induced by γ-rays. A characteristic feature of their action is the formation of clustered
DNA lesions which are difficulty repaired or cannot be repaired at all. Using the immunofluorescence
staining method and confocal microscopy 3D-images of the nuclei of human fibroblasts irradiated with
1Gy of γ-rays (60Co, LET = 0.3 keV/μm) and accelerated 11B ions (LET = 135 keV/μm) were obtained.
Samples were irradiated vertically for the study of the DNA repair kinetics and horizontally (10°) for the
analysis of the formation and structure of clustered DNA double strand breaks (DSB) damage along the
ion track trajectory. In order to quantify the induction and repair of DNA lesions, γH2AX and 53BP1 repair
foci - markers of DNA DSB which form discrete foci at DSB sites - were quantified. Our results show that
irradiation with 11B ions leads to the formation of approximately three-times higher amount of
γH2AX/53BP1 foci as compared to γ-rays. The maximum yield of foci after γ-irradiation is achieved at 1 h
post irradiation (PI) and within 4 h PI most of the foci (~80 %) are eliminated. In cells exposed to 11B ions,
the highest yield of γH2AX/53BP1 foci is observed at 45 min PI. In opposed to γ-rays in cells irradiated by
accelerated 11B ions, within 4 h PI the number of foci decreases just on 55 %, and the elimination of 80 %
379 | P a g e
4 Gy irradiated groups, and DNA recovered from each clone. We confirmed each clone as derived from
single cell by detecting rearrangement patterns of the JH region in the IGH gene. Whole genome
sequencing of DNA from the 7 clones and DNA from the original line was carried out at the Beijing
Genomics Institute by the 90 bp paired-end method using Hiseq2000. After obtaining 90 Gb of raw
sequence data per sample, we mapped them to human reference sequence GRCh37/hg19 by BWA and
analyzed using samtools and in-house programs to identify mutation candidates. We judged the change
of sequences as mutation candidates when observed only in one clone but not observed in other clones,
the original cell line, and the reference sequence. We identified 12 relatively large deletions in control and
irradiated clones consistent with those identified by microarray CGH analyses previously conducted in our
laboratory. Translocation and inversion type mutations were also identified only in the exposed group.
Approximately 20-30 small (<5 bp) indel mutation candidates were also found in control and irradiated
clones. The majority of candidates involved simple repeat sequences such as AAAA to AAA or GCGC to
GCGCGC. Among indels in unique sequences, only from 3-9 candidates per clone were able to be
confirmed as true mutations with Sanger sequencing. We also identified ~1,500 mutation candidates
involving single nucleotide substitutions per clone. Using a stringent filtering (variant frequency >0.3,
depth of coverage > 20, and locating at non-repetitive region for enabling a validation by Sanger
sequencing), the number of the true base substitution mutationswas estimated to be 100-400 per clone.
Based on these data we found that the number of indel- and base-substitution mutations was increased
following irradiation.
(PS7-50) Induction and repair of clustered DNA double strand breaks in human fibroblasts after
1
4
1;2
1;3
irradiation with Boron ions and γ-rays. Lucie Jezkova ; Alla Boreyko ; Tatiana Bulanova ; Martin Falk ;
1;3
1;3
4
4
5
Iva Falkova ; Marie Davidkova ; Stanislav Kozubek ; Evgeny Krasavin ; Elena Kruglyakova ; Olga
2
1;3
1
Valentova ; and Mariia Zadneprianetc , Joint Institute for Nuclear Research, Dubna, Russian Federation ;
2
Institute of Chemical Technology Prague, Prague, Czech Republic ; "Dubna" International University for
3
Nature, Society, and Man, Dubna, Russian Federation ; Institute of Biophysics, Academy of Sciences of the
4
Czech Republic, Brno, Czech Republic ; and Nuclear Physics Institute, Academy of Sciences of the Czech
Republic, Prague, Czech Republic
5
The nature of the damage in DNA caused by the accelerated charged particles significantly differs
from the damage induced by γ-rays. A characteristic feature of their action is the formation of clustered
DNA lesions which are difficulty repaired or cannot be repaired at all. Using the immunofluorescence
staining method and confocal microscopy 3D-images of the nuclei of human fibroblasts irradiated with
1Gy of γ-rays (60Co, LET = 0.3 keV/μm) and accelerated 11B ions (LET = 135 keV/μm) were obtained.
Samples were irradiated vertically for the study of the DNA repair kinetics and horizontally (10°) for the
analysis of the formation and structure of clustered DNA double strand breaks (DSB) damage along the
ion track trajectory. In order to quantify the induction and repair of DNA lesions, γH2AX and 53BP1 repair
foci - markers of DNA DSB which form discrete foci at DSB sites - were quantified. Our results show that
irradiation with 11B ions leads to the formation of approximately three-times higher amount of
γH2AX/53BP1 foci as compared to γ-rays. The maximum yield of foci after γ-irradiation is achieved at 1 h
post irradiation (PI) and within 4 h PI most of the foci (~80 %) are eliminated. In cells exposed to 11B ions,
the highest yield of γH2AX/53BP1 foci is observed at 45 min PI. In opposed to γ-rays in cells irradiated by
accelerated 11B ions, within 4 h PI the number of foci decreases just on 55 %, and the elimination of 80 %
379 | P a g e
