Retrospective Dosimetry by Translocation Analysis

1
Retrospective DosimetrybyTranslocation Analysis

• Lecture
• Module 5

2
Introduction

• Recognized drawback of dicentric and cytokinesis
  block micronucleus (CBMN) assays is that damage
  is unstable and therefore is eliminated from
  peripheral blood lymphocyte pool at rate that
  cell renewal occurs
• It has long been recognized that analysis for
  more persistent types of damage, e.g. stable
  translocations, is needed to address biodosimetry
  for old or long term exposures
• FISH techniques has made this possible

3
Approach before FISH

• To calculate corrected dose taking into account
  the mean life of lymphocytes and exponential loss
  of cells with unstable aberrations
• There are few data which enable reliable
  correction factor to be deduced
• depending on factors such as infections,
  depression of aberration yield probably cannot be
  expressed simply as function of time alone
• Nevertheless, an exponential disappearance rate
  with a half-time of about three years is
  accepted. As a general approximation this seems
  suitable when the sampling delay is long, say
  five or more years

4
Initial dose x exp(-0.693 x i/3). i elapsed
time in years
years Dose (Gy) Dose (Gy)
  2 4
0 2 4
1 1,59 3,17
2 1,26 2,52
3 1,00 2,00
4 0,79 1,59
5 0,63 1,26
6 0,50 1,00
7 0,40 0,79
8 0,32 0,63
9 0,25 0,50
10 0,20 0,40
11 0,16 0,32
12 0,13 0,25
13 0,10 0,20
14 0,08 0,16
15 0,06 0,13
16 0,05 0,10
17 0,04 0,08
18 0,03 0,06
19 0,02 0,05
20 0,02 0,04
After 2 and 4 Gy irradiation expected decrease
in the estimated dose using dicentrics. This
simulation considers an exponential disappearance
rate of lymphocytes carrying unstable aberrations
with a half-time of three years.
5
How valid is 3 years half life value?
This seems to apply only to people with normal
hematology. There is evidence that following high
acute exposure reduction of dicentrics is
biphasic initially fast and then slower.
Persistence of each phase seems to depend on
initial frequency of aberrations
Data from highly irradiated victims of the
Chernobyl accident. Sevankaev et al., Radiat.
Prot. Dosim. 113 152-161 (2005)
6
Another approach is to consider the dilution with
time of undamaged cells entering the circulation,
and to estimate the initial dose using the method
applied for partial irradiations. This is not
feasible after long periods.
This approximation gives realistic results only
in some cases
Data from highly irradiated victims of the
Chernobyl accident. Sevankaev et al., Radiat.
Prot. Dosim. 113 152-161 (2005)
7
After DNA damage by ionizing radiations,
misrepair of broken pieces can produce a
dicentric chromosome, this type of aberration can
easily be visualized using solid stain.
In theory there is the same probability for
radiation to form a dicentric or a reciprocal
translocation. There is experimental evidence
supporting a 11 ratio. Translocations can be
visualized after laborious banded karyotyping or
more easily by FISH
8
FISH
Fluorescence in situ Hybridization (FISH)
techniques using DNA probes that hybridize with
the entire chromosome length produces
multicoloured Chromosome painting
9

• Example accident in Estonia (1)

After the accident the frequency of dicentrics
decreases clearly as time passes
Data from Lindholm, Int.J. Radiat. Biol. 70
647-656 (1996) Lindholm et al., Int.J. Radiat.
Biol. 74 565-571 (1998) Lindholm and Edwards,
Int.J. Radiat. Biol. 80 559-566 (2004)
10
Example accident in Estonia (2)
But the frequency of translocations remained
relatively constant
Data from Lindholm, Int.J. Radiat. Biol. 70
647-656 (1996) Lindholm et al., Int.J. Radiat.
Biol. 74 565-571 (1998) Lindholm and Edwards,
Int.J. Radiat. Biol. 80 559-566 (2004)
11
Main steps of the FISH technique
Cocktail of DNA probes labelled with
fluorochromes
Fixed cells on slides
Denaturation of probe and target
Hybridization and post-hybridization washes
Counterstain with a fluorescent dye, and analysis
using fluorescence microscopy
12
FISH techniques have many applications in
medicine and in fundamental cytogenetics and can
be applied to metaphases and nuclei
Binucleated lymphocytes hybridized with
centrometric specific probes for chromosomes 4
(green and red labelled DNA probes, resulting in
a yellow signal), chromosome 7 (green labelled)
and 18 (red labelled).
Human metaphase with monocoloured painted
chromosomes 1, 4 and 11 labeled with Cy3
(red), centromeres highlighted with a
pancetromeric probe labeled with FITC (green),
and the rest counterstained with DAPI.
13
With range of probes and fluorochrome
combinations now commercially available it is
possible to highlight all chromosomes by method
known as multicolour FISH (mFISH). This permits
full karyotyping and thus scoring all
inter-chromosomal translocations. Centromeres and
telomeres of all chromosomes can be separately
highlighted too.
Left picture using pan-telomeric (red signals)
and pan-centromeric probes (green signals), white
arrows indicate centric-chromosome fragments
lacking telomeric signals at one end (indicating
an incomplete aberration) yellow arrows indicate
two acentric fragments, and the red arrow a
tricentric. Right picture an mFISH karyotype
where each chromosome pair can be analyzed
individually white arrows indicate an exchange
between chromosomes 6 and 11, and the yellow
arrows between 11 and 12.
14
For biological dosimetry purposes most common
technique is to paint several chromosomes with
one or more fluorochromes, and to detect
aberrations between painted and unpainted
genomic material
Human metaphase with coloured painted chromosomes
2 (FITC, green), 4 (Texas Red) and 8
(FITCTexas Red, yellow), and the rest
counterstained with DAPI. An apparently simple
translocation, or two-way translocation
t(Ba),t(Ab) involving chromosome 2 is
observed.
15
Because the aberrations detected are those
between painted and unpainted material,
efficiency in detecting translocations depends on
number of painted chromosomes
Using a mono-coloured cocktail of chromosomes,
and according to the Lucas formula, the
efficiency in detecting bicoloured aberrations is
2.05fp(1-fp). The maximum efficiency will be
with painting 50 of the genome.
16
Routine FISH analysis

• Generally, painting three of larger chromosomes
  (i.e. 1 to 12), representing about 20 of
  genome, leads to about 33 efficiency in
  detecting translocations when single colour is
  used. Percentage of the genome that each cocktail
  paints is related to total genome considering
  the physical lengths of chromosomes

• It is advisable not to include chromosomes 7 or
  14 in probe combinations, as translocations and
  other aberrations involving these chromosomes can
  arise in vivo during immunological development
  and may thus confound quantification of radiation
  effect

• For retrospective biological dosimetry single
  colour FISH for triple cocktail of target
  chromosomes appears to be sufficient. Multiple
  colour painting of triplet increases detection
  efficiency (if chromosomes 1, 4 and 12 are
  highlighted from about 31 to about 34) and
  gives better detection of complex translocations
  that can be encountered following high recent
  exposures

17
Scoring criteria
There is consensus on which metaphases should be
scored. Well-spread metaphases are considered
suitable for scoring if cells appear to be
intact, centromeres are morphologically
detectable and present in all painted
chromosomes, and fluorochrome labelling is
sufficiently bright to detect exchanges between
chromosomes.
Human metaphase with coloured painted chromosomes
1 (FITC, green), 4 (FITCCy3 yellow) and 11
(Cy2, red), centromeres highlighted with
pancentromeric probe labelled with Cy3 (red), and
rest counterstained with DAPI. Simple
translocation, or two-way translocation
t(Bc),t(Cb) involving chromosomes 1 and 11 is
observed.
18
Nomenclature
To describe the chromosome aberrations detected
by painting two specific nomenclature systems
were developed independently and descriptions
based on the conventional terminology of routine
cytogenetic scoring were also used.
Protocol for Aberration Identification and
Nomenclature Terminology (PAINT) was developed to
be purely descriptive of each aberrant painted
object in the metaphase, without cross-reference
to other aberrant objects in the cell. Each
colour is designated by a letter, starting
alphabetically with the counterstain. Capital
letters designate the component that bears a
centromere, and multiple coloured painting is
accommodated by including further letters in the
nomenclature. Tucker et al., Cytogenet Cell
Genet. 68211-221 (1995).
Counterstained
a A
Painted 1 colour
b B
Painted 2 colour
c C
Painted 3 colour
d D
Examples
t(Ba)
t-ins(Abab)
dic(BA)
t(Ab)
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Savage and Simpson (SS) proposed a terminology
comprising numerals and letters describing each
exchange in its entirety. The exchanges were
classified according to the number of chromosomes
(C), the number of arms (A) and the number breaks
(B) involved (CAB families). This so-called SS
system applies only to single paint patterns.
However, it can be used with dual and triple
paint patterns but each painted chromosome has to
be scored in isolation irrespective of the
colours of partners. This nomenclature has
considerable uses in mechanistic studies,
particularly, for example, in understanding
complex rearrangements.
Examples
Number of breaks needed
2
2
2B
The nomenclature contains aberrations from CAB
2/2/2 to 5/5/5, and it is difficult to handle.
So, for retrospective studies is not suitable.
But has a lot of interest for mechanistic studies.
3
2A
2G
Name of the exchange
3
2F
20
A more conventional terminology may be employed
that names translocations as reciprocal or
terminal. Reciprocal has also been called
complete or two-way, and terminal is also called
incomplete or one-way.
Reciprocal
Terminal
Incomplete, or one-way translocations
Complete, or two-way translocation
21
Nomenclature used in practice

• Nowadays most widely used method for recording
  data is to describe each abnormal metaphase as a
  unit using PAINT nomenclature but in slightly
  modified way that considers underlying
  mechanisms of formation of aberrations

• Abbreviations of PAINT system are used but a
  note is made of associations between objects in
  metaphase, thereby incorporating aspects of the
  conventional terminology too

• Chromosome aberrations are classified as simple
  or complex, latter being when three or more
  breaks in two or more chromosomes are needed to
  produce observed abnormality. Aberrations are
  considered complete when all broken pieces are
  rejoined and as incomplete when one or more
  pieces appear unrejoined

22
This metaphase contains a dicentric between two
painted chromosomes and a dicentric between two
unpainted chromosomes. So a dic (BB)ace (b) plus
and dic (AA)ace(a)
dic(BB) ace(b)
dic(AA) ace(a)
23
More examples
Ins(Bab)
Ins(Aba)
24
What is stable? This metaphase contains
dicentric between unpainted chromosomes and
translocation between painted and unpainted
chromosomes. So, dic(AA)ace(a) plus and
t(Ba)t(Ab). It is important to consider that
translocation is stable aberration, but this cell
is unstable due to presence of dicentric and its
acentric
t(Ba) t(Ba)
Dic(AA) ace(a)
25
Dose-Effect Curves
Similar to dicentrics, for dose estimations with
translocations detected by FISH, each laboratory
needs to establish its own curves. Curve should
be made with same FISH probe cocktail that is
routinely used for case investigations. Doing
this removes need to convert to genome
equivalence which could introduce some extra
uncertainty
It is recommended to score all aberrations
detected in entire chromosome set, not just those
affecting the painted material, but also those
affecting unpainted material. This allows one to
establish dose-effect curves for translocations
in stable cells (cells without dicentrics, rings
or acentrics)
26
Dose-Effect Curves
However, it should be pointed that for low-LET
radiations, when calibration curves for
translocations have been constructed taking into
account stable or total cells, there were no
differences in the fitted coefficients if only
apparently simple translocations were considered
Dose effect curves for all simple translocations
observed in all cells, or those only in stable
cells
t(Ba)t(Ab)
two-way translocation
t(Ab)
one-way translocations

t(Ba)
All simple translocations
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Data Handling

• When only translocations between painted and
  unpainted chromosomes material are scored it may
  be necessary to convert observed frequency to
  full genome equivalence
• Conversion is recommended procedure to use when
  data have to be combined or intercomparisons have
  to be made between results from various studies
  where different combinations of whole chromosome
  painting probes have been used

• Assumption, sometimes referred to as Lucas
  formula, is that probability of the involvement
  of particular painted chromosome in aberration is
  proportional to its DNA content. This assumption
  gives reasonable approximation. However, there is
  consensus that when using the (DNA content) in
  Lucas formula, larger chromosomes may tend to be
  overestimated in their participation in simple
  exchange aberrations compared to smaller ones

• Therefore, use in Lucas formula of (DNA
  content)2/3 rather than (DNA content) gives more
  accurate results. Some authors have argued that
  this kind of proportionality could be symptomatic
  of interchanges involving primarily chromatin
  near boundary of chromosome territories

28
Genomic equivalence- painting with one colour
fp is the fraction of the genome covered by the
DNA probes, and 1-fp the remainder unpainted
genome
All possible exchanges are between painted and
painted Can not be detected painted and
unpainted Can be detected unpainted and
painted unpainted and unpainted Can not be
detected Intrachromosomal exchanges, that can
not be detected 0.026. This number depends on
the number of human chromosomes and the relative
DNA content of them.
If the total exchanges is FG, and the the
fraction of all exchanges than can be detected is
Fp then
The genomic equivalent frequency of translocation
is calculated using this formula
29
Example
If chromosome pairs 1, 2 and 4 are painted. Their
respective DNA contents (male) are 0.0828, 0.0804
and 0.0639. 2743. Therefore, fp 0.2271, so
that FP/FG 0.360 This combination of
chromosomes painted is 36 efficient in measuring
bicoloured translocations. Therefore, to obtain
the full genome translocation yield the observed
yield is divided by 0.36
30
Control Level of Translocations (1)

• Control levels of translocations are higher
  than for dicentrics, and to some extent this is
  due to translocations being a persisting type of
  aberration
• It is therefore important to take the
  translocation background into account,
  particularly after low doses, when attempting
  retrospective biodosimetry

31
Control Level of Translocations (2)

• Comprehensive meta-analysis currently provides
  best international database, broken down by age,
  gender, race and smoking habits (Sigurdson et al.
  2008)
• It is clear that age is major factor that
  determines background frequency of translocations

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Age-control relationship
From number of translocations observed in
individual, it is important to subtract the
generic background frequency expected for his or
her age
This is genomic equivalent frequency
Data from Sigurdson et al., Mutat. Res
652112-121 (2008)